Methods of identifying and monitoring mitochondrial dysfunction using monocyte screening

ABSTRACT

The present technology provides methods for detecting and diagnosing diseases and conditions characterized by mitochondrial dysfunction using monocytes as an indicator of the dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase application under 35 U.S.C.§ 371 of International Application No. PCT/US2015/036130, filed Jun. 17,2015 which claims the benefit of and priority to U.S. Application No.62/013,317, filed Jun. 17, 2014, the contents of which are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The present technology relates generally to methods for detecting anddiagnosing diseases and conditions characterized by mitochondrialdysfunction using monocytes as an indicator of the dysfunction.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art.

Many chronic pathological conditions associated with mitochondrialdysfunction such as metabolic syndrome, diabetes, neurodegenerativediseases, and atherosclerosis are associated with an inflammatoryresponse with the release of proinflammatory mediators, particularlycytokines. Monocytes are phagocytic cells that play an important role inthe innate immune system. Once secreted from the bone marrow into theblood, these cells survey the body for sites of inflammation. Onencountering inflammatory stress signals the monocytes must rapidlyactivate and migrate to areas of injury where they can differentiateinto the pro-inflammatory “killer” (M1) or anti-inflammatory “repair”(M2) phenotype. Both human classical and intermediate monocytes haveinflammatory properties that are reminiscent of M1 phenotype, whilenon-classical monocytes display properties similar to M2 phenotype.

In the M1 state, the activated monocyte-macrophage cell undergoes ametabolic switch from oxidative phosphorylation to glycolysis. Thisswitch is important because it provides substrates for biosyntheticprograms, maintains mitochondrial membrane potential and results in ATPproduction within the cell. Inhibition of oxidative phosphorylation alsoincreases reactive oxygen species (ROS) production which exertsbactericidal activities. During the resolution of inflammation, themacrophages transform into the alternatively activated M2 phenotype anda more oxidative phosphorylation phenotype. Thus, the metabolic programsof monocyte/macrophage populations are highly plastic and adapt tofacilitate the changing function of these cells in the inflammatoryprocess. Currently, it is unclear whether early changes in metabolicphenotype associated with exposure to pro-inflammatory conditions can bedetected in the pre-differentiated circulating monocytes.

SUMMARY OF THE PRESENT TECHNOLOGY

There is a need for accurate and sensitive methods that permit theidentification and monitoring of a broad range of pathologicalconditions including but not limited to ischemia, stroke, renal injury,neurodegenerative diseases, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease and Leigh's disease.

In one aspect, the present technology provides a method for identifyinga disease or condition characterized by mitochondrial dysfunction in asubject, comprising (a) assaying the level of a population of activatedmonocytes present in a biological sample obtained from the subject, and(b) comparing the level of the population of activated monocytesobserved in step (a) with the level of a corresponding population ofactivated monocytes observed in a reference sample, wherein the subjectis identified as having a disease or condition characterized bymitochondrial dysfunction if the level of the population of activatedmonocytes present in the biological sample is increased compared to thereference sample.

In some embodiments, the reference sample is a biological sampleobtained from a healthy subject.

In some embodiments, the total count of activated monocytes present inthe biological sample is increased compared to the reference sample. Insome embodiments, the level of classical monocytes (CD14^(high) CD16⁻)is elevated compared to the reference sample. In some embodiments, thelevel of intermediate monocytes (CD14^(high)CD16⁺) is elevated comparedto the reference sample. In a further embodiment, the level ofnon-classical monocytes (CD14^(low)CD16^(high)) is decreased compared tothe reference sample. In some embodiments, the monocytes are circulatingmonocytes. In other embodiments, the monocytes are extravasated from thebloodstream to other tissues.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method foridentifying a disease or condition characterized by mitochondrialdysfunction in a subject, comprising (a) assaying the ratio of differentmonocyte types present in a biological sample obtained from the subject,and (b) comparing the ratio of different monocyte types observed in step(a) with the ratio of corresponding monocyte types observed in areference sample, wherein the subject is identified as having a diseaseor condition characterized by mitochondrial dysfunction if the ratio ofdifferent monocyte types present in the biological sample is alteredcompared to the reference sample.

In some embodiments, the reference sample is a biological sampleobtained from a healthy subject. In some embodiments, the monocytes arecirculating monocytes. In other embodiments, the monocytes areextravasated from the bloodstream to other tissues. In some embodiments,the ratio of activated monocytes to non-classical monocytes is elevatedcompared to the reference sample. In some embodiments, the ratio ofclassical monocytes to non-classical monocytes is elevated compared tothe reference sample. In some embodiments, the ratio of intermediatemonocytes to non-classical monocytes is elevated compared to thereference sample. In certain embodiments, the ratio of classicalmonocytes to intermediate monocytes is elevated compared to thereference sample.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method foridentifying a disease or condition characterized by mitochondrialdysfunction in a subject, comprising (a) assaying at least one biomarkerof mitochondrial physiology of a population of monocytes present in abiological sample obtained from the subject, and (b) comparing thebiomarker of mitochondrial physiology of the population of monocytesobserved in step (a) with the biomarker of mitochondrial physiology of acorresponding population of monocytes observed in a reference sample,wherein the subject is identified as having a disease or conditioncharacterized by mitochondrial dysfunction if the biomarker ofmitochondrial physiology of the population of monocytes present in thebiological sample is altered compared to the reference sample.

In some embodiments, the reference sample is a biological sampleobtained from a healthy subject. In some embodiments, the monocytes arecirculating monocytes. In other embodiments, the monocytes areextravasated from the bloodstream to other tissues.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction results in a disruption in oxidativephosphorylation.

In some embodiments, alterations in the biomarkers of mitochondrialphysiology of monocytes can be determined by assaying levels of one ormore biomarkers of mitochondrial physiology selected from the groupconsisting of lactic acid (lactate) levels, pyruvic acid (pyruvate)levels, lactate/pyruvate ratios, phosphocreatine levels, NADH (NADH+H³⁰)or NADPH (NADPH+H³⁰) levels; NAD or NADP levels; ATP levels; reducedcoenzyme Q (CoQred) levels; oxidized coenzyme Q (CoQox) levels; totalcoenzyme Q (CoQtot) levels; oxidized cytochrome C levels; reducedcytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio;acetoacetate levels; beta-hydroxy butyrate levels;acetoacetate/beta-hydroxy butyrate ratio; 8-hydroxy-2′-deoxyguanosine(8-OHdG) levels; levels of reactive oxygen species; oxygen consumption(VO2), carbon dioxide output (VCO2), and respiratory quotient(VCO2/VO2).

One aspect of the present technology provides a method for evaluatingthe therapeutic efficacy of an aromatic-cationic peptide on a disease orcondition characterized by mitochondrial dysfunction in a subject,comprising (a) assaying the level of a population of activated monocytespresent in a biological sample obtained from the subject, and (b)comparing the level of the population of activated monocytes observed instep (a) with the level of a corresponding population of activatedmonocytes observed in a biological sample obtained from the subjectfollowing administration of a dose of an aromatic-cationic peptide,wherein the aromatic-cationic peptide is identified as having atherapeutic effect on the disease or condition characterized bymitochondrial dysfunction if the level of the population of activatedmonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the level of thepopulation of activated monocytes observed in step (a).

In some embodiments, the aromatic-cationic peptide isPhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the level of classical monocytes (CD14^(high)CD16⁻)in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the level of classicalmonocytes observed in step (a). In some embodiments, the level ofintermediate monocytes (CD14^(high)CD16⁺) in the biological samplefollowing the administration of the aromatic-cationic peptide is reducedcompared to the level of intermediate monocytes observed in step (a). Ina further embodiment, the level of non-classical monocytes(CD14^(low)CD16^(high)) in the biological sample following theadministration of the aromatic-cationic peptide is increased compared tothe level of non-classical monocytes observed in step (a).

In some embodiments, the monocytes are circulating monocytes. In otherembodiments, the monocytes are extravasated from the bloodstream toother tissues.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method forevaluating the therapeutic efficacy of an aromatic-cationic peptide on adisease or condition characterized by mitochondrial dysfunction in asubject, comprising (a) assaying the ratio of different monocyte typespresent in a biological sample obtained from the subject, and (b)comparing the ratio of different monocyte types observed in step (a)with the ratio of corresponding monocyte types observed in a biologicalsample obtained from the subject following administration of a dose ofan aromatic-cationic peptide, wherein the aromatic-cationic peptide isidentified as having a therapeutic effect on the disease or conditioncharacterized by mitochondrial dysfunction if the ratio of differentmonocyte types in the biological sample following the administration ofthe aromatic-cationic peptide is altered compared to the ratio ofdifferent monocyte types observed in step (a).

In some embodiments, the monocytes are circulating monocytes. In otherembodiments, the monocytes are extravasated from the bloodstream toother tissues.

In some embodiments, the ratio of activated monocytes to non-classicalmonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio of activatedmonocytes to non-classical monocytes observed in step (a). In someembodiments, the ratio of classical monocytes to non-classical monocytesin the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio of classicalmonocytes to non-classical monocytes observed in step (a). In someembodiments, the ratio of intermediate monocytes to non-classicalmonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio ofintermediate monocytes to non-classical monocytes observed in step (a).

In some embodiments, the ratio of classical monocytes to intermediatemonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio of classicalmonocytes to intermediate monocytes observed in step (a).

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method forevaluating the therapeutic efficacy of an aromatic-cationic peptide on adisease or condition characterized by mitochondrial dysfunction in asubject, comprising (a) assaying at least one biomarker of mitochondrialphysiology of a population of monocytes present in a biological sampleobtained from the subject, and (b) comparing the biomarker ofmitochondrial physiology of the population of monocytes observed in step(a) with the biomarker of mitochondrial physiology of a correspondingpopulation of monocytes observed in a biological sample obtained fromthe subject following administration of a dose of an aromatic-cationicpeptide, wherein the aromatic-cationic peptide is identified as having atherapeutic effect on the disease or condition characterized bymitochondrial dysfunction if the biomarker of mitochondrial physiologyof the population of monocytes in the biological sample following theadministration of the aromatic-cationic peptide is similar to thebiomarker of mitochondrial physiology of a corresponding population ofmonocytes in a reference sample. In some embodiments, the referencesample is a biological sample obtained from a healthy subject.

In some embodiments, the aromatic-cationic peptide isPhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof. In some embodiments, the monocytes arecirculating monocytes. In other embodiments, the monocytes areextravasated from the bloodstream to other tissues.

In some embodiments, alterations in the biomarkers of mitochondrialphysiology of monocytes can be determined using assays that measuredisruption in oxidative phosphorylation. In some embodiments, disruptionin oxidative phosphorylation is determined using assays that measureCoQ10 levels, uncoupling ratio, net routine flux control ratio, leakflux control ratio or phosphorylation respiratory control ratio. In someembodiments, alterations in the biomarkers of mitochondrial physiologyof monocytes can be determined by measuring alterations in the levels ofone or more biomarkers of mitochondrial physiology in a sample ofmonocytes. In some embodiments, biomarkers of mitochondrial physiologyare selected from the group consisting of consisting of lactic acid(lactate) levels, pyruvic acid (pyruvate) levels, lactate/pyruvateratios, phosphocreatine levels, NADH (NADH+H³⁰) or NADPH (NADPH+H³⁰)levels; NAD or NADP levels; ATP levels; reduced coenzyme Q (CoQred)levels; oxidized coenzyme Q (CoQox) levels; total coenzyme Q (CoQtot)levels; oxidized cytochrome C levels; reduced cytochrome C levels;oxidized cytochrome C/reduced cytochrome C ratio; acetoacetate levels;beta-hydroxy butyrate levels; acetoacetate/beta-hydroxy butyrate ratio;8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels of reactive oxygenspecies; oxygen consumption (VO2), carbon dioxide output (VCO2), andrespiratory quotient (VCO2/VO2).

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In some embodiments, alterations in the biomarkers of mitochondrialphysiology are determined using high throughput bioenergetics platforms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the effect of D-Arg-2′6′Dmt-Lys-Phe-NH₂ on parameters ofmonocyte mitochondrial function over time. FIG. 1B shows the effect ofsaline on parameters of monocyte mitochondrial function over time. FIG.1C shows a comparison of ATP production, maximal respiration, andrespiratory capacity between the saline and D-Arg-2′6′Dmt-Lys-Phe-NH₂treatment groups. Dogs with induced heart failure received a single4-hour intravenous infusion of saline or D-Arg-2′6′Dmt-Lys-Phe-NH₂.

FIG. 2 illustrates the effect that various dysfunctions can have onbiochemistry and biomarkers of mitochondrial physiology. It alsoindicates the physical effect (such as a disease symptom or other effectof the dysfunction) typically associated with a given dysfunction. Itshould be noted that any of the biomarkers of mitochondrial physiologylisted in the figure, in addition to biomarkers of mitochondrialphysiology enumerated elsewhere, can also be used to detect modulation,enhancement, or normalization by the compositions of the presenttechnology in a sample of monocytes. RQ=respiratory quotient; BMR=basalmetabolic rate; HR (CO)=heart rate (cardiac output); T=body temperature(preferably measured as core temperature); AT=anaerobic threshold;pH=blood pH venous and/or arterial).

DETAILED DESCRIPTION

General

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the present technology are described below invarious levels of detail in order to provide a substantial understandingof the present technology.

While the aromatic-cationic peptides described herein can occur and canbe used as the neutral (non-salt) peptide, the description is intendedto embrace all salts of the peptide described herein, as well as methodsof using such salts of the peptides. In one embodiment, the salts of thearomatic-cationic peptides comprise pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts which can beadministered as drugs or pharmaceuticals to humans and/or animals andwhich, upon administration, retain at least some of the biologicalactivity of the free compound (neutral compound or non-salt compound).The desired salt of a basic peptide may be prepared by methods known tothose of skill in the art by treating the compound with an acid.Examples of inorganic acids include, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, andphosphoric acid. Examples of organic acids include, but are not limitedto, formic acid, acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, sulfonic acids, and salicylic acid. Salts of basic peptides withamino acids, such as aspartate salts and glutamate salts, can also beprepared. The desired salt of an acidic peptide can be prepared bymethods known to those of skill in the art by treating the compound witha base. Examples of inorganic salts of acidic peptides include, but arenot limited to, alkali metal and alkaline earth salts, such as sodiumsalts, potassium salts, magnesium salts, and calcium salts; ammoniumsalts; and aluminum salts. Examples of organic salts of acidic peptidesinclude, but are not limited to, procaine, dibenzylamine,N-ethylpiperidine, N,N′-dibenzylethylenediamine, and triethylaminesalts. Salts of acidic peptides with amino acids, such as lysine salts,can also be prepared. The present technology also includes allstereoisomers and geometric isomers of the peptides of the presenttechnology, including diastereomers, enantiomers, and cis/trans (E/Z)isomers. The present technology also includes mixtures of stereoisomersand/or geometric isomers in any ratio, including, but not limited to,racemic mixtures.

I Definitions

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this present technologybelongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs are compounds that have the samebasic chemical structure as a naturally-occurring amino acid, i.e., anα-carbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, e.g., homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally-occurring amino acid. Amino acidmimetics are chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally-occurring amino acid. Amino acids canbe referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “biological sample” refers to a sample from thesubject, and includes any bodily fluids, exudates, tissues or cells.Non-limiting examples include blood, plasma, serum, urine, tears,sputum, stool, saliva, nasal swabs, cells such as, but not limited toperipheral blood mononuclear cells (PBMCs), leukocytes, and tissuesamples (e.g., biopsy samples). Samples can be fresh, frozen, orotherwise treated or preserved for evaluation by the methods disclosedherein. In some embodiments, levels and mitochondrial physiologicalactivity of monocytes are determined by assaying a biological samplefrom a subject using techniques that are well-known in the art.

As used herein, the term “biomarkers of mitochondrial physiology” refersto one or more physiological parameters that can be used to assess thefrequency, output and regulation of distinct chemical and/or physicalprocesses occurring within the mitochondria of monocytes and areselected from the group consisting of lactic acid (lactate) levels,pyruvic acid (pyruvate) levels, lactate/pyruvate ratios, phosphocreatinelevels, NADH (NADH+H³⁰) or NADPH (NADPH+H³⁰) levels; NAD or NADP levels;ATP levels; reduced coenzyme Q (CoQred) levels; oxidized coenzyme Q(CoQox) levels; total coenzyme Q (CoQtot) levels; oxidized cytochrome Clevels; reduced cytochrome C levels; oxidized cytochrome C/reducedcytochrome C ratio; acetoacetate levels; beta-hydroxy butyrate levels;acetoacetate/beta-hydroxy butyrate ratio; 8-hydroxy-2′-deoxyguanosine(8-OHdG) levels; levels of reactive oxygen species; oxygen consumption(VO2), carbon dioxide output (VCO2), respiratory quotient (VCO2/VO2) andany additional physiological parameters assessed by high resolutionrespirometry (e.g., Oxygraph-2k (OROBOROS)) and high throughputbioenergetics platforms such as XF Analyzers (Seahorse Biosciences).

The terms “circulating blood” or ‘whole blood’, which are used asequivalents herein, refer to the blood within the closed cardiovascularsystem of vertebrates. This comprises the blood in arteries (arterialblood), in veins (venous blood) as well as capillaries, venules andportal systems. Circulating blood cells are the cellular components ofblood, consisting of red blood cells, white blood cells, and platelets,which are found within the circulating pool of blood and not sequesteredwithin the lymphatic system, spleen, liver, or bone marrow. The term‘whole blood’ encompasses both peripheral blood, found in the systemiccirculation, and central blood, present in the pulmonary and coronarycirculation.

The term “circulating monocytes” refers to monocytes that are present inthe (peripheral or central) blood and thus part of the circulation (andnot migrated into tissue). A “sample of monocytes” as used herein refersto a sample wherein the majority of cells are monocytes. In certainembodiments, the sample consists essentially of monocytes, or itconsists exclusively of monocytes. Since only monocytes give rise tomacrophages or dendritic cells (the myeloid lineage), and it isdesirable that the sample represents this lineage, it is particularlyenvisaged that the sample contains as little other blood cells asfeasible. Thus, it is envisaged that no (or virtually none, or as littleas feasible) other leukocytes are present in the sample, that no (orvirtually none, or as little as feasible) granulocytes are present inthe sample, that no (or virtually none, or as little as feasible) otherPBMCs are present in the sample, that no (or virtually none, or aslittle as feasible) lymphocytes (T cells, B cells, NK cells) are presentin the sample.

According to particular embodiments, the sample does not consist of amixed monocyte-lymphocyte population. In other words, the sample is nota population of peripheral blood mononuclear cells. According toalternative embodiments, the sample does not contain lymphocytes. Thus,a sample of monocytes can be interpreted as a sample of isolatedmonocytes. In other words, although circulating blood containsmonocytes, according to particular embodiments, it does not fulfill thedefinition of a sample of monocytes. In some embodiments, to obtain asample of monocytes from a blood sample, a further separation orisolation step is needed. Typically, by way of example but not by way oflimitation, this is done by density gradient separation (to isolate thePBMC from the rest of the blood constituents) followed bymarker-assisted separation; but a one-step isolation procedure (bymarker(s) only) can be applied as well. For example, in humans, one canseparate the CD14-expressing cells in peripheral blood from thenon-CD14-expressing cells to obtain a sample of monocytes as envisagedherein. Monocytes can be further divided into subpopulations, forinstance depending on expression of particular receptors (e.g., but notlimited to, CD16).

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in adisease or condition or one or more symptoms associated with a diseaseor condition characterized by mitochondrial dysfunction. In the contextof therapeutic or prophylactic applications, the amount of a compositionadministered to the subject will depend on the type and severity of thedisease and on the characteristics of the individual, such as generalhealth, age, sex, body weight and tolerance to drugs. It will alsodepend on the degree, severity and type of disease. The skilled artisanwill be able to determine appropriate dosages depending on these andother factors. As discussed herein, active agents such asaromatic-cationic peptide compositions can also be administered incombination with one or more additional therapeutic compounds. In themethods described herein, the active agents (e.g., an aromatic-cationicpeptide) may be administered to a subject having one or more signs orsymptoms of a disease or condition characterized by mitochondrialdysfunction. For example, a “therapeutically effective amount” of theactive agents (e.g., an aromatic-cationic peptide) is meant levels inwhich the physiological effects of a disease or condition characterizedby mitochondrial dysfunction are, at a minimum, ameliorated. Atherapeutically effective amount can be given in one or moreadministrations. In some embodiments, signs, symptoms or complicationsof a disease or condition characterized by mitochondrial dysfunctioninclude, but are not limited to: increased levels of activatedmonocytes, poor growth, loss of muscle coordination, muscle weakness,neurological deficit, seizures, autism, autistic spectrum, autistic-likefeatures, learning disabilities, heart disease, liver disease, kidneydisease, gastrointestinal disorders, severe constipation, diabetes,increased risk of infection, thyroid dysfunction, adrenal dysfunction,autonomic dysfunction, confusion, disorientation, memory loss, failureto thrive, poor coordination, sensory (vision, hearing) problems,reduced mental functions, disease of the organ, dementia, respiratoryproblems, hypoglycemia, apnea, lactic acidosis, seizures, swallowingdifficulties, developmental delays, movement disorders (dystonia, musclespasms, tremors, chorea), stroke, and brain atrophy. In someembodiments, an “effective amount” of a compound is an amount of thecompound sufficient to modulate, normalize, or enhance one or morebiomarkers of mitochondrial physiology (where modulation, normalization,and enhancement are defined below) in cells, such as monocytes.

As used herein, “enhancement” of, or to “enhance,” biomarkers ofmitochondrial physiology means to intentionally change the level of oneor more biomarkers of mitochondrial physiology in cells, such asmonocytes, that are present in a biological sample away from either thenormal value, or the value before enhancement, in order to achieve abeneficial or desired effect. For example, enhancement can be ofbeneficial effect in a subject suffering from a disease or conditioncharacterized by mitochondrial dysfunction, in that normalizing abiomarker of mitochondrial physiology may not achieve the optimumoutcome for the subject; in such cases, enhancement of one or morebiomarkers of mitochondrial physiology can be beneficial, for example,higher-than-normal levels of ATP, or lower-than normal levels of lacticacid (lactate) can be beneficial to such a subject.

As used herein, the term “heart failure” encompasses all forms of heartfailure, including but not limited to, e.g., “congestive heart failure”(CHF), “chronic heart failure,” and “acute heart failure.” As usedherein, the term encompasses both sporadic and genetic forms of heartfailure. As is known in the art, heart failure is typicallycharacterized by abnormally low cardiac output in which the heart isunable to pump blood at an adequate rate or in adequate volume. When theheart is unable to adequately pump blood to the rest of the body, orwhen one or more of the heart valves becomes stenotic or otherwiseincompetent, blood can back up into the lungs, causing the lungs tobecome congested with fluid. If this backward flow occurs over anextended period of time, heart failure can result. Typical symptoms ofheart failure include shortness of breath (dyspnea), fatigue, weakness,difficulty breathing when lying flat, and swelling of the legs, anklesor abdomen (edema). Causes of heart failure may be related to variousdisorders including coronary artery disease, systemic hypertension,cardiomyopathy or myocarditis, congenital heart disease, abnormal heartvalves or valvular heart disease, severe lung disease, diabetes, severeanemia hyperthyroidism, arrhythmia or dysrhythmia and myocardialinfarction. The primary signs of congestive heart failure arecardiomegaly (enlarged heart), tachypnea (rapid breathing; occurs in thecase of left side failure) and hepatomegaly (enlarged liver; occurs inthe case of right side failure).

As used herein, the term “hypertensive cardiomyopathy” refers to acondition characterized by a weakened heart caused by the effects ofhypertension (high blood pressure). Over time, uncontrolled hypertensioncauses weakness of the heart muscle. As hypertensive cardiomyopathyworsens, it can lead to congestive heart failure. Early symptoms ofhypertensive cardiomyopathy include cough, weakness, and fatigue.Additional symptoms of hypertensive cardiomyopathy include leg swelling,weight gain, difficulty breathing when lying flat, increasing shortnessof breath with activity, and waking in the middle of the night short ofbreath.

As used herein, the term “ischemia” refers to a decrease in the bloodsupply to the tissue and is followed by “reperfusion,” a suddenperfusion of oxygen into the deprived tissue.

As used herein, an “isolated” or “purified” polypeptide or peptide issubstantially free of cellular material or other contaminatingpolypeptides from the cell or tissue source from which the agent isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. For example, an isolatedaromatic-cationic peptide would be free of materials that wouldinterfere with diagnostic or therapeutic uses of the agent. Suchinterfering materials may include enzymes, hormones and otherproteinaceous and nonproteinaceous solutes.

As used herein, the term “disease or condition characterized bymitochondrial dysfunction” refers to a disease or condition selectedfrom the group consisting of ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis. In someembodiments, the disease or condition characterized by mitochondrialdysfunction manifests as a result of genetic factors. In otherembodiments, the disease or condition characterized by mitochondrialdysfunction is induced by non-genetic factors.

The term “monocytes” as used herein refers to a particular type of whiteblood cells (leukocytes) that are part of the innate immune system ofvertebrates. Monocytes are normally produced by the bone marrow fromhematopoietic stem cell precursors called monoblasts. They circulate inthe bloodstream for about one to three days and then typically move intotissues throughout the body. Monocytes constitute between three to eightpercent of the leukocytes in the blood (reference values in healthyadult humans), and are the largest corpuscle in blood. Once extravasatedfrom the bloodstream to other tissues, they will differentiate intotissue resident macrophages or dendritic cells. Monocytes play multipleroles in immune function such as: (1) replenishing resident macrophagesunder normal states, and (2) rapidly migrating to sites of infection ormetabolic stress in tissues in response to inflammation signals. Thereare at least three types of monocytes in human blood: a) the classicalmonocyte, which is characterized by high level expression of the CD14cell surface receptor (CD14⁺⁺ CD16⁻ monocyte), b) the non-classicalmonocyte, which shows low level expression of CD14 and high levelexpression of the CD16 receptor (CD14⁺CD16⁺⁺ monocyte), and c) theintermediate monocyte with high level expression of CD14 and low levelexpression of CD16 (CD14⁺⁺CD16⁺ monocytes). In humans, CD14 isconsidered a marker of the monocyte lineage. So, at least in humans,‘monocytes’ can be considered equivalent to CD14-expressing cells thatcirculate in the bloodstream (the latter property distinguishing themfrom dendritic cells and macrophages). Although virtually allCD14-expressing cells in peripheral blood will be monocytes, furtherdifferentiation using other markers or cell size can be made todistinguish monocytes from other cell types.

As used herein, the “modulation” of, or to “modulate,” a biomarker ofmitochondrial physiology means to change the level of the biomarker ofmitochondrial physiology in monocytes that are present in a biologicalsample towards a desired value, or to change the level of the biomarkerof mitochondrial physiology in a desired direction (e.g., increase ordecrease). Modulation can include, but is not limited to, normalizationand enhancement as defined herein.

As used herein, the term “net charge” refers to the balance of thenumber of positive charges and the number of negative charges carried bythe amino acids present in the aromatic-cationic peptide. In thisspecification, it is understood that net charges are measured atphysiological pH. The naturally occurring amino acids that arepositively charged at physiological pH include L-lysine, L-arginine, andL-histidine. The naturally occurring amino acids that are negativelycharged at physiological pH include L-aspartic acid and L-glutamic acid.

As used herein, “normalization” of, or to “normalize,” a biomarker ofmitochondrial physiology means changing the level of the biomarker ofmitochondrial physiology in cells, such as monocytes, that are presentin a biological sample, from a pathological value towards a normalvalue, where the normal value of the biomarker of mitochondrialphysiology can be 1) the level of the biomarker of mitochondrialphysiology in a healthy person or subject, or 2) a level of thebiomarker of mitochondrial physiology that alleviates one or moreundesirable symptoms in the person or subject. That is, to normalize abiomarker of mitochondrial physiology which is depressed in a diseasestate means to increase the level of the biomarker of mitochondrialphysiology towards the normal (healthy) value or towards a value whichalleviates an undesirable symptom; to normalize a biomarker ofmitochondrial physiology which is elevated in a disease state means todecrease the level of the biomarker of mitochondrial physiology towardsthe normal (healthy) value or towards a value which alleviates anundesirable symptom.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, “preventing” or “prevention” of a disease or conditioncharacterized by mitochondrial dysfunction refers to one or morecompounds that, in a statistical sample, reduces the occurrence of thedisorder or condition in the treated sample relative to an untreatedcontrol sample, or delays the onset or reduces the severity of one ormore symptoms of the disorder or condition relative to the untreatedcontrol sample. As used herein, preventing a disease or conditioncharacterized by mitochondrial dysfunction includes preventing oxidativedamage or preventing mitochondrial permeability transitioning, therebypreventing or ameliorating the harmful effects of the disruption ofmitochondrial oxidative phosphorylation.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the terms “subject” “individual,” or “patient” can be anindividual organism, a vertebrate, a mammal, or a human.

A “synergistic therapeutic effect” refers to a greater-than-additivetherapeutic effect which is produced by a combination of at least twotherapeutic agents, and which exceeds that which would otherwise resultfrom individual administration of the therapeutic agents alone. Forexample, lower doses of one or both of the therapeutic agents may beused in treating a disease, resulting in increased therapeutic efficacyand decreased side-effects.

As used herein, the term “therapeutic use” of the compounds discussedherein is defined as using one or more of the active agents discussedherein to treat or suppress a disease, as defined herein.

As used herein, the term “transient ischemic attack (TIA)” refers toperiods when blood flow to a part of the brain is interrupted for abrief period of time. TIAs last from a few minutes to 1-2 hours, and mayoccur again at a later time. The symptoms of TIA are the same as thesymptoms of a stroke, and include sudden vertigo or dizziness, suddenchanges in alertness, sudden changes in sensory perception (includingtouch, pain, temperature, pressure, hearing, and taste), memory loss,difficulty swallowing, drooping of the face, inability to recognizeobjects or people, lack of bladder or bowel control, lack ofcoordination, loss of vision in one or both eyes, weakness, numbness ortingling on one side of the body etc.

“Treating” or “treatment” as used herein covers the treatment of adisease or disorder described herein, in a subject, such as a human, andincludes: (i) inhibiting a disease or disorder, i.e., arresting itsdevelopment; (ii) relieving a disease or disorder, i.e., causingregression of the disorder; (iii) slowing progression of the disorder;and/or (iv) inhibiting, relieving, or slowing progression of one or moresymptoms of the disease or disorder. For example, a subject issuccessfully “treated” for a disease or disorder characterized bymitochondrial dysfunction if, after receiving a therapeutic amount ofthe active agents (e.g., an aromatic-cationic peptide) according to themethods described herein, the subject shows observable and/or measurablereduction in the disruption of mitochondrial oxidative phosphorylation.It is also to be appreciated that the various modes of treatment orprevention of medical diseases and conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved.

II Aromatic-Cationic Peptides of the Present Technology

The aromatic-cationic peptides of the present technology arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes. The aromatic-cationic peptidestypically include a minimum of three amino acids or a minimum of fouramino acids, covalently joined by peptide bonds. The maximum number ofamino acids present in the aromatic-cationic peptides is about twentyamino acids covalently joined by peptide bonds. Suitably, the maximumnumber of amino acids is about twelve, or about nine, or about six.

In some aspects, the present technology provides an aromatic-cationicpeptide or a pharmaceutically acceptable salt thereof such as acetatesalt or trifluoroacetate salt. In some embodiments, the peptidecomprises at least one net positive charge; a minimum of three aminoacids; a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and

a relationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1.

In some embodiments, the peptide comprises the amino acid sequencePhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′-Dmt-Lys-Phe-NH₂. In someembodiments, the peptide comprises one or more of the peptides of TableA:

TABLE A Phe-Arg-D-His-Asp Met-Tyr-D-Lys-Phe-Arg Phe-D-Arg-HisTyr-D-Arg-Phe-Lys-NH₂ 2′6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′6′-Dmt-D-Arg-PheOrn-NH₂ 2′6′-Dmt-D-Cit-Phe Lys-NH₂ Phe-D-Arg-2′6′-Dmt-Lys-NH₂2′6′-Dmt-D-Arg-Phe-Ahp-NH₂ H-Phe-D-Arg-Phe-Lys-Cys-NH₂2′6′-Dmp-D-Arg-2′6′-Dmt-Lys-NH₂ 2′6′-Dmp-D-Arg-Phe-Lys-NH₂Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg Lys-Gln-Tyr-D-Arg-Phe-TrpD-Arg-2′6′-Dmt-Lys-Trp-NH₂ D-Arg-Trp-Lys-Trp-NH₂D-Arg-2′6′-Dmt-Lys-Phe-Met-NH₂ D-Arg-2′6′-Dmt-Lys(N^(a)Me)-Phe-NH₂D-Arg-2′6′-Dmt-Lys-Phe(NMe)—NH₂ D-Arg-2′6′-Dmt-Lys(N^(a)Me)-Phe(NMe)—NH₂D-Arg(N^(a)Me)-2′6′-Dmt(NMe)-Lys(N^(a)Me)-Phe(NMe)—NH₂D-Arg-2′6′-Dmt-Lys-Phe-Lys-Trp-NH₂D-Arg-2′6′-Dmt-Lys-2′6′-Dmt-Lys-Trp-NH₂D-Arg-2′6′-Dmt-Lys-Phe-Lys-Met-NH₂D-Arg-2′6′-Dmt-Lys-2′6′-Dmt-Lys-Met-NH₂D-Arg-2′6′-Dmt-Lys-Phe-Sar-Gly-Cys-NH₂D-Arg-Ψ[CH₂—NH]2′6′-Dmt-Lys-Phe-NH₂ D-Arg-2′6′-Dmt-Ψ[CH₂—NH]Lys-Phe-NH₂D-Arg-2′6′-Dmt-LysΨ[CH₂—NH]Phe-NH₂D-Arg-2′6′-Dmt-Ψ[CH₂—NH]Lys-Ψ[CH₂—NH]Phe-NH₂ Lys-D-Arg-Tyr-NH₂D-Tyr-Trp-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂ Tyr-His-D-Gly-MetTyr-D-Arg-Phe-Lys-Glu-NH₂ Met-Tyr-D-Arg-Phe-Arg-NH₂D-His-Glu-Lys-Tyr-D-Phe-Arg Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysLys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysAsp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheTyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-PhePhe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysGlu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met- NH₂Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D- Arg-GlyD-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-PheHis-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-AspThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ 2′,6′-dimethyltyrosine (2′6′-Dmt);dimethyltyrosine (Dmt)

In one embodiment, the aromatic-cationic peptide is defined by FormulaA:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the peptide is defined by Formula B:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹and R¹² are all hydrogen; and n is 4. In another embodiment, R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² are methyl;R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the aromatic-cationic peptides of the presenttechnology have a core structural motif of alternating aromatic andcationic amino acids. For example, the peptide may be a tetrapeptidedefined by any of Formulas C to F set forth below:Aromatic-Cationic-Aromatic-Cationic  (Formula C)Cationic-Aromatic-Cationic-Aromatic  (Formula D)Aromatic-Aromatic-Cationic-Cationic  (Formula E)Cationic-Cationic-Aromatic-Aromatic  (Formula F)wherein, aromatic is a residue selected from the group consisting of:Phe (F), Tyr (Y), Trp (W), and Cyclohexylalanine (Cha); and Cationic isa residue selected from the group consisting of: Arg (R), Lys (K),Norleucine (Nle), and 2-amino-heptanoic acid (Ahe).

The peptides disclosed herein may be formulated as pharmaceuticallyacceptable salts. The term “pharmaceutically acceptable salt” means asalt prepared from a base or an acid which is acceptable foradministration to a patient, such as a mammal (e.g., salts havingacceptable mammalian safety for a given dosage regime). However, it isunderstood that the salts are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a peptide contains both a basic moiety, such asan amine, pyridine or imidazole, and an acidic moiety such as acarboxylic acid or tetrazole, zwitterions may be formed and are includedwithin the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic,lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids(e.g., acetic, butyric, formic, propionic and trifluoroacetic acids),amino acids (e.g., aspartic and glutamic acids), aromatic carboxylicacids (e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic,hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g.,o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylicand 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylicacids (e.g., fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(e.g., benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like. In someembodiments, the salt is an acetate salt. Additionally or alternatively,in other embodiments, the salt is a trifluoroacetate salt.

The aromatic-cationic peptides of the present technology disclosedherein may be synthesized by any of the methods well known in the art.Suitable methods for chemically synthesizing the protein include, forexample, liquid phase and solid phase synthesis, and those methodsdescribed by Stuart and Young in Solid Phase Peptide Synthesis, SecondEdition, Pierce Chemical Company (1984), and in Methods Enzymol., 289,Academic Press, Inc, New York (1997). Recombinant peptides may begenerated using conventional techniques in molecular biology, proteinbiochemistry, cell biology, and microbiology, such as those described inCurrent Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed.(1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed.(1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic AcidHybridization, Hames & Higgins, Eds. (1985); Transcription andTranslation, Hames & Higgins, Eds. (1984); Animal Cell Culture,Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986);Perbal, A Practical Guide to Molecular Cloning; the series, Meth.Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors forMammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu,Eds., respectively.

Aromatic-cationic peptide precursors may be made by either chemical(e.g., using solution and solid phase chemical peptide synthesis) orrecombinant syntheses known in the art. Precursors of e.g., amidatedaromatic-cationic peptides of the present technology may be made in likemanner. In some embodiments, recombinant production is believedsignificantly more cost effective. In some embodiments, precursors areconverted to active peptides by amidation reactions that are also knownin the art. For example, enzymatic amidation is described in U.S. Pat.No. 4,708,934 and European Patent Publications 0 308 067 and 0 382 403.Recombinant production can be used for both the precursor and the enzymethat catalyzes the conversion of the precursor to the desired activeform of the aromatic-cationic peptide. Such recombinant production isdiscussed in Biotechnology, Vol. 11 (1993) pp. 64-70, which furtherdescribes a conversion of a precursor to an amidated product. Duringamidation, a keto-acid such as an alpha-keto acid, or salt or esterthereof, wherein the alpha-keto acid has the molecular structureRC(O)C(O)OH, and wherein R is selected from the group consisting ofaryl, a C₁-C₄ hydrocarbon moiety, a halogenated or hydroxylated C₁-C₄hydrocarbon moiety, and a C₁-C₄ carboxylic acid, may be used in place ofa catalase co-factor. Examples of these keto acids include, but are notlimited to, ethyl pyruvate, pyruvic acid and salts thereof, methylpyruvate, benzoyl formic acid and salts thereof, 2-ketobutyric acid andsalts thereof, 3-methyl-2-oxobutanoic acid and salts thereof, and 2-ketoglutaric acid and salts thereof.

In some embodiments, the production of the recombinant aromatic-cationicpeptide may proceed, for example, by producing glycine-extendedprecursor in E. coli as a soluble fusion protein withglutathione-S-transferase. An α-amidating enzyme catalyzes conversion ofprecursors to active aromatic-cationic peptide. That enzyme isrecombinantly produced, for example, in Chinese Hamster Ovary (CHO)cells as described in the Biotechnology article cited above. Otherprecursors to other amidated peptides may be produced in like manner.Peptides that do not require amidation or other additionalfunctionalities may also be produced in like manner. Other peptideactive agents are commercially available or may be produced bytechniques known in the art.

The peptides optionally contain one or more non-naturally occurringamino acids. Optimally, the peptide has no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g., methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are suitably resistant orinsensitive to common proteases. Examples of non-naturally occurringamino acids that are resistant or insensitive to proteases include thedextrorotatory (D-) form of any of the above-mentioned naturallyoccurring L-amino acids, as well as L- and/or D-non-naturally occurringamino acids. The D-amino acids do not normally occur in proteins,although they are found in certain peptide antibiotics that aresynthesized by means other than the normal ribosomal protein syntheticmachinery of the cell. As used herein, the D-amino acids are consideredto be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, or less than four, or less than three, or less than twocontiguous L-amino acids recognized by common proteases, irrespective ofwhether the amino acids are naturally or non-naturally occurring.Optimally, the peptide has only D-amino acids, and no L-amino acids. Ifthe peptide contains protease sensitive sequences of amino acids, atleast one of the amino acids is a non-naturally-occurring D-amino acid,thereby conferring protease resistance. An example of a proteasesensitive sequence includes two or more contiguous basic amino acidsthat are readily cleaved by common proteases, such as endopeptidases andtrypsin. Examples of basic amino acids include arginine, lysine andhistidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, or a minimum of two net positivecharges, or a minimum of three net positive charges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

TABLE 5 EXEMPLARY PEPTIDES 2′,6′-Dmp-D-Arg-2′,6′-Dmt-Lys-NH₂2′,6′-Dmp-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Arg-PheOrn-NH₂2′,6′-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)—NH₂2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Cit-PheLys-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheArg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D- Arg-GlyAsp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-Arg-2′,6′-Dmt-Lys-Phe-NH₂D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂ D-His-Glu-Lys-Tyr-D-Phe-ArgD-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH₂D-Tyr-Trp-Lys-NH₂Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met- NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp. Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂ Lys-D-Arg-Tyr-NH₂ Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂ Met-Tyr-D-Arg-Phe-Arg-NH₂Met-Tyr-D-Lys-Phe-Arg Phe-Arg-D-His-Asp Phe-D-Arg-2′,6′-Dmt-Lys-NH₂Phe-D-Arg-His Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Phe-D-Arg-Phe-Lys-NH₂Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrThr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysTyr-D-Arg-Phe-Lys-Glu-NH₂ Tyr-D-Arg-Phe-Lys-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Tyr-His-D-Gly-MetVal-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂

In one embodiment, the peptides have mu-opioid receptor agonist activity(i.e., they activate the mu-opioid receptor). Peptides which havemu-opioid receptor agonist activity are typically those peptides whichhave a tyrosine residue or a tyrosine derivative at the N-terminus(i.e., the first amino acid position). Suitable derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′,6′-dimethyltyrosine (2′6′-Dmt);3′,5′-dimethyltyrosine (3′5′Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltyrosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂. Tyr-D-Arg-Phe-Lys-NH₂has a net positive charge of three, contributed by the amino acidstyrosine, arginine, and lysine and has two aromatic groups contributedby the amino acids phenylalanine and tyrosine. The tyrosine ofTyr-D-Arg-Phe-Lys-NH₂ can be a modified derivative of tyrosine such asin 2′,6′-dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-Phe-Lys-NH₂. 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ has a molecularweight of 640 and carries a net three positive charge at physiologicalpH. 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ readily penetrates the plasma membraneof several mammalian cell types in an energy-independent manner (Zhao etal., J. Pharmacol Exp Ther., 304:425-432, 2003).

Alternatively, in other instances, the aromatic-cationic peptide doesnot have mu-opioid receptor agonist activity. For example, duringlong-term treatment, such as in a chronic disease state or condition,the use of an aromatic-cationic peptide that activates the mu-opioidreceptor may be contraindicated. In these instances, the potentiallyadverse or addictive effects of the aromatic-cationic peptide maypreclude the use of an aromatic-cationic peptide that activates themu-opioid receptor in the treatment regimen of a human patient or othermammal. Potential adverse effects may include sedation, constipation andrespiratory depression. In such instances an aromatic-cationic peptidethat does not activate the mu-opioid receptor may be an appropriatetreatment. Peptides that do not have mu-opioid receptor agonist activitygenerally do not have a tyrosine residue or a derivative of tyrosine atthe N-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂.Alternatively, the N-terminal phenylalanine can be a derivative ofphenylalanine such as 2′,6′-dimethylphenylalanine (2′6′-Dmp). In oneembodiment, the amino acid sequence of 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ isrearranged such that Dmt is not at the N-terminus. An example of such anaromatic-cationic peptide that does not have mu-opioid receptor agonistactivity has the formula D-Arg-2′6′-Dmt-Lys-Phe-NH₂.

Suitable substitution variants of the peptides listed herein includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group are referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group are generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that activate mu-opioid receptors include, but arenot limited to, the aromatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs with Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ TyrD-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH-dns NH₂ 2′6′Dmt D-Arg PheLys-NH(CH₂)₂—NH-atn NH₂ 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit PheLys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′DmtD-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg PheAhp(2-aminoheptanoic acid) NH₂ Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′DmtD-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg TyrDap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys PheDab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-LysTyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys TyrDap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′DmtD-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt LysNH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′DmtD-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg PheatnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap PheArg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides that do not activate mu-opioid receptors include,but are not limited to, the aromatic-cationic peptides shown in Table 7.

TABLE 7 Peptide Analogs Lacking Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 6 and 7 may be in eitherthe L- or the D-configuration.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc., New York (1997).

Synthesis of the Aromatic-Cationic Peptides of the Present Technology

The aromatic-cationic peptides useful in the methods of the presenttechnology may be chemically synthesized by any of the methods wellknown in the art. Suitable methods for synthesizing the protein include,for example, those described by Stuart and Young in “Solid Phase PeptideSynthesis,” Second Edition, Pierce Chemical Company (1984), and in“Solid Phase Peptide Synthesis,” Methods Enzymol. 289, Academic Press,Inc, New York (1997).

In practicing the present technology, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

III Use of Monocytes in Diagnostic Methods

The cellular components of peripheral blood include red blood cells,white blood cells, and platelets. White blood cells, or leukocytes, arecells of the immune system involved in defending the body against bothinfectious disease and foreign materials. Five different and diversetypes of leukocytes exist: neutrophils, basophils, and eosinophils(together the granulocytes or polymorphonuclear leukocytes), lymphocytesand monocytes (together the a granulocytes, mononuclear leukocytes orperipheral blood mononuclear cells (PBMCs)). The three major types oflymphocyte (all from the lymphoid lineage) are T cells and B cells (themajor cellular components of the adaptive immune response) and naturalkiller (NK) cells. Monocytes are derived from the myeloid lineage andare part of the innate immune system and are able to migrate quickly toa site of inflammation in the body. Circulating monocytes present inwhole blood can give rise to macrophages and dendritic cells.

Currently, the most common approaches to measure cellular bioenergeticshave been to isolate mitochondria from biopsies, peripheral blood cellsor develop fibroblast cell lines from patient samples. The drawback ofthese approaches is that PBMC do not have high levels of mitochondria,biopsies are generally invasive and painful, and in the case offibroblasts the passage of the cells may alter their bioenergetics.

Levels and/or biomarkers of mitochondrial physiology of circulatingmonocytes are altered from the very earliest stages of a disease orcondition. Many such diseases or conditions are associated withmitochondrial dysfunction. In some embodiments, the disease or conditionis an inflammatory disease or a disease with an inflammatory componentsuch as ischemia, stroke, renal injury, neurodegenerative diseases,atherosclerosis, metabolic syndrome, acute myocardial infarction, heartfailure, ischemia reperfusion, ureteral obstruction, diabeticnephropathy, diabetes, Leber's Heredity Optic Neuropathy (LHON),Dominant Optic Atrophy (DOA), Barth's syndrome, POLG disease, andLeigh's disease. In some embodiments, the neurodegenerative disease isAlzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), Huntington'sdisease, Friedreich's ataxia, or Multiple Sclerosis. In principle, foreach of these inflammatory diseases a specific monocyte signature can bederived, comprising at least one physiological parameter that isdifferentially regulated in monocytes. These observed physiologicaldifferences of circulating monocytes are caused by factors associatedwith the disease or condition characterized by mitochondrialdysfunction, such as increased oxidative stress or defects inmitochondrial permeability transition. The differences in the levelsand/or biomarkers of mitochondrial physiology of circulating monocytescan be used as accurate and sensitive methods for detecting anddiagnosing diseases or conditions characterized by mitochondrialdysfunction in a subject. The biomarkers of mitochondrial physiologydescribed herein can be evaluated in a minimally invasive way, as only asmall sample of (typically peripheral) blood is required. Moreover, asubset of parameters in the monocyte physiological profile can be usedto discriminate between patients with different types and/or severitiesof diseases or conditions characterized by mitochondrial dysfunction.This allows stratification of patients, and may help guide decisions onsuitable therapies.

In one aspect, the present technology provides a method for identifyinga disease or condition characterized by mitochondrial dysfunction in asubject, comprising (a) assaying the level of a population of activatedmonocytes present in a biological sample obtained from the subject, and(b) comparing the level of the population of activated monocytesobserved in step (a) with the level of a corresponding population ofactivated monocytes observed in a reference sample, wherein the subjectis identified as having a disease or condition characterized bymitochondrial dysfunction if the level of the population of activatedmonocytes present in the biological sample is increased compared to thereference sample.

In some embodiments, the reference sample is a biological sampleobtained from a healthy subject.

In some embodiments, the total count of activated monocytes present inthe biological sample is increased compared to the reference sample. Insome embodiments, the level of classical monocytes (CD14^(high)CD16⁻) iselevated compared to the reference sample. In some embodiments, thelevel of intermediate monocytes (CD14^(high)CD16⁺) is elevated comparedto the reference sample. In a further embodiment, the level ofnon-classical monocytes (CD14^(low)CD16^(high)) is decreased compared tothe reference sample. In some embodiments, the monocytes are circulatingmonocytes. In other embodiments, the monocytes are extravasated from thebloodstream to other tissues.

Suitable techniques to determine overall monocyte count as well as thedistribution of the different types of monocytes include flow cytometryor FACS. In particular, FACS analysis can be used to determine if thereis an expansion or alteration of a given subpopulation of monocytes,thereby unmasking a signal that may otherwise be undetectable in a wholeblood sample. Such alterations serve as a predictor of a disease stateor condition characterized by mitochondrial dysfunction. Many of thesetechniques can also be done using alternatives to classical antibodies,e.g. nanobodies (single domain antibodies, developed by Ablynx),alphabodies (single-chain, triple-stranded coiled coil proteins,developed by Complix) or other protein-binding molecules.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In some embodiments, the biological sample is a blood sample. In certainembodiments, the monocytes are isolated from a blood sample (e.g.,peripheral or central blood sample). In a particular embodiment, theblood sample is obtained no more than 12 hours, no more than 10 hours,no more than 8 hours, no more than 6 hours, no more than 4 hours or nomore than 2 hours prior to starting the isolation of the monocytes fromthe blood sample.

In another embodiment, the present technology provides a method foridentifying a disease or condition characterized by mitochondrialdysfunction in a subject, comprising (a) assaying the ratio of differentmonocyte types present in a biological sample obtained from the subject,and (b) comparing the ratio of different monocyte types observed in step(a) with the ratio of corresponding monocyte types observed in areference sample, wherein the subject is identified as having a diseaseor condition characterized by mitochondrial dysfunction if the ratio ofdifferent monocyte types present in the biological sample is alteredcompared to the reference sample.

In some embodiments, the reference sample is a biological sampleobtained from a healthy subject. In some embodiments, the monocytes arecirculating monocytes. In other embodiments, the monocytes areextravasated from the bloodstream to other tissues. In some embodiments,the ratio of activated monocytes to non-classical monocytes is elevatedcompared to the reference sample. In some embodiments, the ratio ofclassical monocytes to non-classical monocytes is elevated compared tothe reference sample. In some embodiments, the ratio of intermediatemonocytes to non-classical monocytes is elevated compared to thereference sample. In certain embodiments, the ratio of classicalmonocytes to intermediate monocytes is elevated compared to thereference sample.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method foridentifying a disease or condition characterized by mitochondrialdysfunction in a subject, comprising (a) assaying at least one biomarkerof mitochondrial physiology of a population of monocytes present in abiological sample obtained from the subject, and (b) comparing thebiomarker of mitochondrial physiology of the population of monocytesobserved in step (a) with the biomarker of mitochondrial physiology of acorresponding population of monocytes observed in a reference sample,wherein the subject is identified as having a disease or conditioncharacterized by mitochondrial dysfunction if the biomarker ofmitochondrial physiology of the population of monocytes present in thebiological sample is altered compared to the reference sample.

In some embodiments, the reference sample is a biological sampleobtained from a healthy subject. In some embodiments, the monocytes arecirculating monocytes. In other embodiments, the monocytes areextravasated from the bloodstream to other tissues.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction results in a disruption in oxidativephosphorylation. A sample of monocytes can be evaluated for disruptionin oxidative phosphorylation using assays well known in the art.Typically, the methods will be performed in vitro. By way of example,but not by way of limitation, a disruption in oxidative phosphorylationis determined by assays that measures levels of coenzyme Q₁₀ (CoQ10). Insome embodiments, disruption in oxidative phosphorylation is determinedby assays that measure OXPHOS capacity by the uncoupling ratio. In someembodiments, disruption in oxidative phosphorylation is determined byassays that measure the net routine flux control ratio. In someembodiments, disruption in oxidative phosphorylation is determined byassays that measure leak flux control ratio. In some embodiments,disruption in oxidative phosphorylation is determined by assays thatmeasure the phosphorylation respiratory control ratio.

Uncoupling ratio (UCR) is an expression of the respiratory reservecapacity and indicates the OXPHOS capacity of the cells. In someembodiments, UCR is defined as Cr_(u)/Cr. Cr_(u) is the maximum rate ofoxygen utilization (Oxygen flux) produced when mitochondria arechemically uncoupled using FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone). FCCP titration must be performed since theconcentration of FCCP required to produce maximum oxygen utilizationvaries among different cell lines. Once the maximum oxygen utilizationis reached, further increases in FCCP inhibit oxygen utilization byoxidative phosphorylation. In some embodiments, Cr represents oxygenutilization by the cells during a normal cellular respiration withexcess substrates.

In some embodiments, the Net Routine Flux Control Ratio (Cr/Cr_(u)) isthe inverse of the UCR. In some embodiments, this value assesses howclose routine respiration operates to the respiratory capacity ofoxidative phosphorylation.

In some embodiments, the Respiratory Control Ratio (RCR) is defined asCr_(u)/Cr_(o). Cr_(u) is defined above. Cr_(o)=Respiration afterinhibition of Complex V (ATP synthase) by oligomycin. In someembodiments, this ratio allows assessment of uncoupling and OXPHOSdysfunction.

In some embodiments, the Leak Flux Control Ratio is determined byCr_(o)/Cr_(u). In some embodiments, this parameter is the inverse of RCRand represents proton leak with inhibition of ADP phosphorylation byoligomycin.

In some embodiments, the Phosphorylation Respiratory Control Ratio(RCRp) is defined as (Cr−Cr_(o))/Cr_(u) (or 1/UCR−1/RCR). In someembodiments, the RCRp is an index which expressesphosphorylation-related respiration (Cr−Cr_(o)) as a function ofrespiratory capacity (Cr_(u)). In some embodiments, the RCRp remainsconstant, if partial uncoupling is fully compensated by an increasedroutine respiration rate and a constant rate of oxidativephosphorylation is maintained. In some embodiments, if the respiratorycapacity declines without effect on the rate of oxidativephosphorylation; in some embodiments, the RCRp increases, whichindicates that a higher proportion of the maximum capacity is activatedto drive ATP synthesis. In some embodiments, the RCRp declines to zeroin either fully uncoupled cells or in cells under complete metabolicarrest.

In another embodiment, the methods of the present technology can be usedto monitor biomarkers of mitochondrial physiology in a sample ofmonocytes to assess the presence or absence of a disease or conditioncharacterized by mitochondrial dysfunction in a subject.

In some embodiments, alterations in the biomarkers of mitochondrialphysiology of monocytes can be detected by assaying the levels of one ormore biomarkers of mitochondrial physiology selected from the groupconsisting of lactic acid (lactate) levels, pyruvic acid (pyruvate)levels, lactate/pyruvate ratios, phosphocreatine levels, NADH (NADH+H³⁰)or NADPH (NADPH+H³⁰) levels; NAD or NADP levels; ATP levels; reducedcoenzyme Q (CoQred) levels; oxidized coenzyme Q (CoQox) levels; totalcoenzyme Q (CoQtot) levels; oxidized cytochrome C levels; reducedcytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio;acetoacetate levels; beta-hydroxy butyrate levels;acetoacetate/beta-hydroxy butyrate ratio; 8-hydroxy-2′-deoxyguanosine(8-OHdG) levels; levels of reactive oxygen species; oxygen consumption(VO2), carbon dioxide output (VCO2), and respiratory quotient(VCO2/VO2). In some embodiments, lactic acid (lactate) levels can bedetermined by assaying lactic acid (lactate) levels in whole blood,plasma, cerebrospinal fluid, or cerebral ventricular fluid. In someembodiments, pyruvic acid (pyruvate) levels can be determined byassaying pyruvic acid (pyruvate) levels in whole blood, plasma,cerebrospinal fluid, or cerebral ventricular fluid. In some embodiments,lactate/pyruvate ratios can be determined by assaying lactate/pyruvateratios in whole blood, plasma, cerebrospinal fluid, or cerebralventricular fluid.

Several of these biomarkers of mitochondrial physiology are discussed inmore detail as follows. It should be emphasized that, while certainbiomarkers of mitochondrial physiology are discussed and enumeratedherein, the present technology is not limited to modulation,normalization or enhancement of only these enumerated biomarkers ofmitochondrial physiology in a sample of monocytes.

Pyruvate, a product of the anaerobic metabolism of glucose, is removedby reduction to lactic acid in an anaerobic setting or by oxidativemetabolism, which is dependent on a functional mitochondrial respiratorychain. Dysfunction of the respiratory chain may lead to inadequateremoval of lactate and pyruvate from the circulation and elevatedlactate/pyruvate ratios are observed in mitochondrial cytopathies (seeScriver C R, The Metabolic and Molecular Bases of Inherited Disease, 7thed., New York: McGraw-Hill, Health Professions Division, 1995; andMunnich et al., J. Inherit. Metab. Dis. 15(4):448-55 (1992)). Bloodlactate/pyruvate ratio (Chariot et al., Arch. Pathol. Lab. Med.118(7):695-7 (1994)) is, therefore, widely used as a noninvasive testfor detection of mitochondrial cytopathies (see again Scriver C R, TheMetabolic and Molecular Bases of Inherited Disease, 7th ed., New York:McGraw-Hill, Health Professions Division, 1995; and Munnich et al., J.Inherit. Metab. Dis. 15(4):448-55 (1992)) and toxic mitochondrialmyopathies (Chariot et al., Arthritis Rheum. 37(4):583-6 (1994)).Changes in the redox state of mitochondria can be investigated bymeasuring the arterial ketone body ratio(acetoacetate/3-hydroxybutyrate:AKBR) (Ueda et al., J. Cardiol.29(2):95-102 (1997)). Urinary excretion of 8-hydroxy-2′-deoxyguanosine(8-OHdG) often has been used as a biomarker to assess the extent ofrepair of ROS-induced DNA damage in both clinical and occupationalsettings (Erhola et al., FEBS Lett. 409(2):287-91 (1997); Honda et al.,Leuk. Res. 24(6):461-8 (2000); Pilger et al., Free Radic. Res.35(3):273-80 (2001); Kim et al. Environ Health Perspect 112(6):666-71(2004)).

Lactic acid (lactate) levels: Mitochondrial dysfunction typicallyresults in abnormal levels of lactic acid, as pyruvate levels increaseand pyruvate is converted to lactate to maintain capacity forglycolysis. Mitochondrial dysfunction can also result in abnormal levelsof NADH+H³⁰, NADPH+H³⁰, NAD, or NADP, as the reduced nicotinamideadenine dinucleotides are not efficiently processed by the respiratorychain. Lactate levels can be measured by taking samples of appropriatebodily fluids such as whole blood, plasma, or cerebrospinal fluid. Usingmagnetic resonance, lactate levels can be measured in virtually anyvolume of the body desired, such as the brain. Measurement of cerebrallactic acidosis using magnetic resonance in patients is described inKaufmann et al., Neurology 62(8): 1297 (2004). Whole blood, plasma, andcerebrospinal fluid lactate levels can be measured by commerciallyavailable equipment such as the YSI 2300 STAT Plus Glucose & LactateAnalyzer (YSI Life Sciences, Ohio).

NAD, NADP, NADH and NADPH levels: Measurement of NAD, NADP, NADH(NADH+H³⁰) or NADPH (NADPH+H³⁰) can be measured by a variety offluorescent, enzymatic, or electrochemical techniques, e.g., theelectrochemical assay described in US 2005/0067303.

Oxygen consumption (vO₂ or VO2), carbon dioxide output (vCO₂ or VCO2),and respiratory quotient (VCO2/VO2): vO₂ is usually measured eitherwhile resting (resting vO₂) or at maximal exercise intensity (vO₂ max).Optimally, both values will be measured. Measurement of both forms ofvO₂ is readily accomplished using standard equipment from a variety ofvendors, e.g., Korr Medical Technologies, Inc. (Salt Lake City, Utah).VCO2 can also be readily measured, and the ratio of VCO2 to VO2 underthe same conditions (VCO2/VO2, either resting or at maximal exerciseintensity) provides the respiratory quotient (RQ).

Oxidized Cytochrome C, reduced Cytochrome C, and ratio of oxidizedCytochrome C to reduced Cytochrome C: Cytochrome C parameters, such asoxidized cytochrome C levels (Cyt C_(ox)), reduced cytochrome C levels(Cyt C_(red)), and the ratio of oxidized cytochrome C/reduced cytochromeC ratio (Cyt C_(ox))/(Cyt C_(red)), can be measured by in vivo nearinfrared spectroscopy. See, e.g., Rolfe, P., “In vivo near-infraredspectroscopy,” Annu. Rev. Biomed. Eng. 2:715-54 (2000) and Strangman etal., “Non-invasive neuroimaging using near-infrared light” Biol.Psychiatry 52:679-93 (2002).

Similarly, tests for normal and abnormal values of pyruvic acid(pyruvate) levels, lactate/pyruvate ratio, ATP levels, anaerobicthreshold, reduced coenzyme Q (CoQ^(red)) levels, oxidized coenzyme Q(CoQ^(ox)) levels, total coenzyme Q (CoQ^(tot)) levels, oxidizedcytochrome C levels, reduced cytochrome C levels, oxidized cytochromeC/reduced cytochrome C ratio, acetoacetate levels, β-hydroxy butyratelevels, acetoacetate/β-hydroxy butyrate ratio,8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, and levels of reactiveoxygen species are known in the art and can be used to derive ametabolic signature for a biological sample (e.g., monocytes). For thepurposes of the present technology, modulation, normalization, orenhancement of biomarker of mitochondrial physiology includesmodulation, normalization, or enhancement of anaerobic threshold inmonocytes.

In some embodiments, alterations in the biomarkers of mitochondrialphysiology of monocytes can be evaluated using magnetic resonancespectroscopy (MRS). MRS has been useful in the diagnoses ofmitochondrial cytopathy by demonstrating elevations in cerebrospinalfluid (CSF) and cortical white matter lactate using proton MRS (1H-MRS)(Kaufmann et al., Neurology 62(8):1297-302 (2004)). Phosphorous MRS(31P-MRS) has been used to demonstrate low levels of corticalphosphocreatine (PCr) (Matthews et al., Ann. Neurol. 29(4):435-8(1991)), and a delay in PCr recovery kinetics following exercise inskeletal muscle (Matthews et al., Ann. Neurol. 29(4):435-8 (1991);Barbiroli et al., J. Neurol. 242(7):472-7 (1995); Fabrizi et al., J.Neurol. Sci. 137(1):20-7 (1996)). A low skeletal muscle PCr has alsobeen confirmed in patients with mitochondrial cytopathy by directbiochemical measurements.

Additionally, high-resolution respirometry (HRR) of monocytes offersensitive diagnostic tests of integrated mitochondrial function usingstandard cell culture techniques and small needle tissue biopsies.Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols foranalysis of oxidative phosphorylation can aid in the dissection ofmitochondrial respiratory control and the pathophysiology ofmitochondrial diseases. Respiratory states are defined in functionalterms to account for the network of metabolic interactions in complexSUIT protocols with stepwise modulation of coupling and substratecontrol. A regulated degree of intrinsic uncoupling is a hallmark ofoxidative phosphorylation, whereas pathological and toxicologicaldyscoupling is evaluated as a mitochondrial defect. The non-coupledstate of maximum respiration is experimentally induced by titration ofestablished uncouplers (FCCP, DNP), to collapse the proton gradientacross the mitochondrial inner membrane and measure the capacity of theelectron transfer system (ETS, open-circuit operation of respiration).Intrinsic uncoupling and dyscoupling are evaluated as the flux controlratio between non-phosphorylating LEAK respiration (electron flowcoupled to proton pumping to compensate for proton leaks) and ETScapacity. If OXPHOS capacity (maximally ADP stimulated oxygen flux) isless than ETS capacity, the phosphorylation system contributes to fluxcontrol. Physiological Complex I+II substrate combinations supportmaximum ETS and OXPHOS capacities, due to the additive effect ofmultiple electron supply pathways converging at the Q-junction.Substrate control with electron entry separately through Complex I(pyruvate+malate or glutamate+malate) or Complex II (succinate+rotenone)restricts ETS capacity and artificially enhances flux control upstreamof the Q-cycle, providing diagnostic information on specific branches ofthe ETS. In some embodiments, HRR measurements are accompanied by thefluorometric detection of reactive oxygen species (ROS; oxidativestress), ATP and Ca²⁺ production, pH and mitochondrial membranepotential using established fluorescent dyes. A detailed description ofHRR is provided in Gnaiger E (2012), Mitochondr Physiol Network 17.18.OROBOROS MiPNet Publications, Innsbruck: 64 pp, herein incorporated byreference in its entirety.

In some embodiments, the mitochondrial function of monocytes is assessedby simultaneously measuring the basal oxygen consumption, glycolysisrates, ATP production, and respiratory capacity of a sample of monocytesin a single experimental set up. By way of example, XF Analyzers(Seahorse Biosciences) perform cellular bioenergetics assays bycombining an electro-optical instrument with ‘smart plastic’ cartridgesthat enable the real-time measurement of cellular bioenergetics in anon-invasive, high throughput multi-well (e.g., 96-well) microplateformat. By incorporating automated compound addition and solid-statefluorescence sensors in a microplate format, such platforms determine invitro oxygen consumption rate (OCR), and extracellular acidificationrate (ECAR), so as to assess cellular functions such as oxidativephosphorylation, glycolysis, and fatty acid oxidation. High throughputbioenergetics platforms can be used to study respiratory malfunction inmultiple disease states such as ischemia, stroke, renal injury,atherosclerosis, metabolic syndrome, acute myocardial infarction, heartfailure, ischemia reperfusion, ureteral obstruction, diabeticnephropathy, diabetes, Leber's Heredity Optic Neuropathy (LHON),Dominant Optic Atrophy (DOA), Barth's syndrome, POLG disease, Leigh'sdisease, and neurodegenerative diseases such as Alzheimer's disease,Amyotrophic Lateral Sclerosis (ALS), Friedreich's ataxia, Huntington'sdisease or Multiple Sclerosis. Respiratory abnormalities in monocytesmay arise as a result of environmental insult, mitochondrial DNA ornuclear DNA mutation.

In some embodiments, the high throughput bioenergetics platformsfacilitate rapid detection of monocyte responses to substrates,inhibitors, and other perturbants. In another embodiment, monocytes areassayed via high throughput bioenergetics platforms in order to identifysubjects that have or are suspected of having a disease or conditioncharacterized by mitochondrial dysfunction. In some embodiments, thesehigh throughput bioenergetics platforms permit the testing of multipleconditions per assay well.

Alterations in the levels and/or biomarkers of mitochondrial physiologyof monocytes (compared to that observed in a healthy subject) can occuras a result of transient events such as tissue degeneration, stroke, orcardiovascular disease. In certain embodiments, the alteredphysiological profile of isolated monocytes permits the identificationof such transient events. In other embodiments, the alteredphysiological profile of isolated monocytes provides an estimate of thefrequency at which such transient events occur. In some embodiments, thealtered physiological profile of isolated monocytes provides informationon the timing of such transient events. The methods disclosed herein canthus be used to classify the extent/severity of the damage to thesubject, which is subsequently useful in identifying the proper courseof treatment.

IV Use of Monocytes to Evaluate the Therapeutic Effect ofAromatic-Cationic Peptides of the Present Technology

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the biological effect of a specificaromatic-cationic peptide of the present technology and whether itsadministration is indicated for treatment. In various embodiments, invitro assays can be performed with representative animal models, todetermine if a given therapeutic (e.g., an aromatic-cationic peptide)exerts the desired effect in reducing disruption of mitochondrialfunction, such as disruption of OXPHOS. Compounds for use in therapy canbe tested in suitable animal model systems including, but not limited torats, mice, chicken, cows, monkeys, rabbits, and the like, prior totesting in human subjects. Similarly, for in vivo testing, any of theanimal model system known in the art can be used prior to administrationto human subjects.

One aspect of the present technology provides a method for evaluatingthe therapeutic efficacy of an aromatic-cationic peptide on a disease orcondition characterized by mitochondrial dysfunction in a subject,comprising (a) assaying the level of a population of activated monocytespresent in a biological sample obtained from the subject, and (b)comparing the level of the population of activated monocytes observed instep (a) with the level of a corresponding population of activatedmonocytes observed in a biological sample obtained from the subjectfollowing administration of a dose of an aromatic-cationic peptide,wherein the aromatic-cationic peptide is identified as having atherapeutic effect on the disease or condition characterized bymitochondrial dysfunction if the level of the population of activatedmonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the level of thepopulation of activated monocytes observed in step (a).

In some embodiments, the aromatic-cationic peptide isPhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the level of classical monocytes (CD14^(high)CD16⁻)in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the level of classicalmonocytes observed in step (a). In some embodiments, the level ofintermediate monocytes (CD14^(high)CD16⁺) in the biological samplefollowing the administration of the aromatic-cationic peptide is reducedcompared to the level of intermediate monocytes observed in step (a). Ina further embodiment, the level of non-classical monocytes(CD14^(low)CD16^(high)) in the biological sample following theadministration of the aromatic-cationic peptide is increased compared tothe level of non-classical monocytes observed in step (a).

In some embodiments, the monocytes are circulating monocytes. In otherembodiments, the monocytes are extravasated from the bloodstream toother tissues.

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method forevaluating the therapeutic efficacy of an aromatic-cationic peptide on adisease or condition characterized by mitochondrial dysfunction in asubject, comprising (a) assaying the ratio of different monocyte typespresent in a biological sample obtained from the subject, and (b)comparing the ratio of different monocyte types observed in step (a)with the ratio of corresponding monocyte types observed in a biologicalsample obtained from the subject following administration of a dose ofan aromatic-cationic peptide, wherein the aromatic-cationic peptide isidentified as having a therapeutic effect on the disease or conditioncharacterized by mitochondrial dysfunction if the ratio of differentmonocyte types in the biological sample following the administration ofthe aromatic-cationic peptide is altered compared to the ratio ofdifferent monocyte types observed in step (a).

In some embodiments, the monocytes are circulating monocytes. In otherembodiments, the monocytes are extravasated from the bloodstream toother tissues.

In some embodiments, the ratio of activated monocytes to non-classicalmonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio of activatedmonocytes to non-classical monocytes observed in step (a). In someembodiments, the ratio of classical monocytes to non-classical monocytesin the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio of classicalmonocytes to non-classical monocytes observed in step (a). In someembodiments, the ratio of intermediate monocytes to non-classicalmonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio ofintermediate monocytes to non-classical monocytes observed in step (a).

In some embodiments, the ratio of classical monocytes to intermediatemonocytes in the biological sample following the administration of thearomatic-cationic peptide is reduced compared to the ratio of classicalmonocytes to intermediate monocytes observed in step (a).

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In another embodiment, the present technology provides a method forevaluating the therapeutic efficacy of an aromatic-cationic peptide on adisease or condition characterized by mitochondrial dysfunction in asubject, comprising (a) assaying at least one biomarker of mitochondrialphysiology of a population of monocytes present in a biological sampleobtained from the subject, and (b) comparing the biomarker ofmitochondrial physiology of the population of monocytes observed in step(a) with the biomarker of mitochondrial physiology of a correspondingpopulation of monocytes observed in a biological sample obtained fromthe subject following administration of a dose of an aromatic-cationicpeptide, wherein the aromatic-cationic peptide is identified as having atherapeutic effect on the disease or condition characterized bymitochondrial dysfunction if the biomarker of mitochondrial physiologyof the population of monocytes in the biological sample following theadministration of the aromatic-cationic peptide is similar to thebiomarker of mitochondrial physiology of a corresponding population ofmonocytes in a reference sample. In some embodiments, the referencesample is a biological sample obtained from a healthy subject.

In some embodiments, the aromatic-cationic peptide isPhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof. In some embodiments, the monocytes arecirculating monocytes. In other embodiments, the monocytes areextravasated from the bloodstream to other tissues.

In some embodiments, alterations in the biomarkers of mitochondrialphysiology of monocytes can be determined using assays that measuredisruption in oxidative phosphorylation. In some embodiments, disruptionin oxidative phosphorylation is determined using assays that measureCoQ10 levels, uncoupling ratio, net routine flux control ratio, leakflux control ratio or phosphorylation respiratory control ratio. In someembodiments, alterations in the biomarkers of mitochondrial physiologyof monocytes can be determined by measuring alterations in the levels ofone or more biomarkers of mitochondrial physiology in a sample ofmonocytes. In some embodiments, biomarkers of mitochondrial physiologyare selected from the group consisting of consisting of lactic acid(lactate) levels, pyruvic acid (pyruvate) levels, lactate/pyruvateratios, phosphocreatine levels, NADH (NADH+H³⁰) or NADPH (NADPH+H³⁰)levels; NAD or NADP levels; ATP levels; reduced coenzyme Q (CoQred)levels; oxidized coenzyme Q (CoQox) levels; total coenzyme Q (CoQtot)levels; oxidized cytochrome C levels; reduced cytochrome C levels;oxidized cytochrome C/reduced cytochrome C ratio; acetoacetate levels;beta-hydroxy butyrate levels; acetoacetate/beta-hydroxy butyrate ratio;8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels of reactive oxygenspecies; oxygen consumption (VO2), carbon dioxide output (VCO2), andrespiratory quotient (VCO2/VO2).

In some embodiments, the disease or condition characterized bymitochondrial dysfunction is ischemia, stroke, renal injury,neurodegenerative disease, atherosclerosis, metabolic syndrome, acutemyocardial infarction, heart failure, ischemia reperfusion, ureteralobstruction, diabetic nephropathy, diabetes, Leber's Heredity OpticNeuropathy (LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLGdisease, or Leigh's disease. In some embodiments, the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.

In some embodiments, the methods described herein use isolated monocytesto detect the amelioration, suppression or prevention of a disease orcondition characterized by mitochondrial dysfunction in a subject thathas been administered effective amounts of a therapeutic agent such asan aromatic-cationic peptide of the present technology.

In some embodiments, isolated monocytes are useful in detecting adecrease in intracellular ROS (reactive oxygen species) and an increasein cell survival in a subject that has been administered effectiveamounts of a therapeutic agent such as an aromatic-cationic peptide ofthe present technology.

In some embodiments, isolated monocytes are useful in detecting anincrease in cell viability in a subject that has been administeredeffective amounts of a therapeutic agent such as an aromatic-cationicpeptide of the present technology.

In some embodiments, isolated monocytes are useful in detecting adecrease in the percentage of cells showing increased caspase activityin a subject that has been administered effective amounts of atherapeutic agent such as an aromatic-cationic peptide of the presenttechnology.

In some embodiments, isolated monocytes are useful in detecting areduction in the rate of ROS accumulation in a subject that has beenadministered effective amounts of a therapeutic agent such as anaromatic-cationic peptide of the present technology.

In some embodiments, isolated monocytes are useful in detectingsuppression of mitochondrial depolarization and ROS accumulation in asubject that has been administered effective amounts of a therapeuticagent such as an aromatic-cationic peptide of the present technology.

In some embodiments, isolated monocytes are useful in detectinginhibition of lipid peroxidation in a subject that has been administeredeffective amounts of a therapeutic agent such as an aromatic-cationicpeptide of the present technology.

In some embodiments, isolated monocytes are useful in detecting adecrease in apoptosis in a subject that has been administered effectiveamounts of a therapeutic agent such as an aromatic-cationic peptide ofthe present technology.

In some embodiments, isolated monocytes are useful in detecting areduction in the disruption in oxidative phosphorylation in a subjectthat has been administered effective amounts of a therapeutic agent suchas an aromatic-cationic peptide of the present technology.

Use of Monocytes to Detect the Modulation of Biomarkers of MitochondrialPhysiology in Response to Treatment

The methods of the present technology can be used in subjects orpatients to detect the modulation of one or more biomarkers ofmitochondrial physiology in a sample of monocytes in response totreatment with an active agent (e.g., an aromatic-cationic peptide).Modulation of biomarkers of mitochondrial physiology can be done tonormalize biomarkers of mitochondrial physiology in a subject, or toenhance biomarkers of mitochondrial physiology in a subject.

In one embodiment, the biomarker levels are modulated to a value withinabout 2 standard deviations of the value in a healthy subject. Inanother embodiment, the biomarker levels are modulated to a value withinabout 1 standard deviation of the value in a healthy subject. In anotherembodiment, the biomarker levels in a subject are changed by at leastabout 15% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 20% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 30% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 40% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 50% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 75% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 90% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).In another embodiment, the biomarker levels are changed by at leastabout 100% above or below the level in the subject prior toadministration of the active agent (e.g., an aromatic-cationic peptide).

Normalization of one or more biomarkers of mitochondrial physiology isdefined as either restoring the level of one or more such biomarkers ofmitochondrial physiology to normal or near-normal levels in a subjectwhose levels of one or more biomarkers of mitochondrial physiology showpathological differences from normal levels (i.e., levels in a healthysubject), or to change the levels of one or more biomarkers ofmitochondrial physiology to alleviate pathological symptoms in asubject. Depending on the nature of the biomarker of mitochondrialphysiology, such levels may show measured values either above or below anormal value. For example, a pathological lactate level is typicallyhigher than the lactate level in a normal (i.e., healthy) person, and adecrease in the level may be desirable. A pathological ATP level istypically lower than the ATP level in a normal (i.e., healthy) person,and an increase in the level of ATP may be desirable. Accordingly,normalization of biomarkers of mitochondrial physiology can involverestoring the level of biomarkers of mitochondrial physiology to withinabout at least two standard deviations of normal in a subject, or towithin about at least one standard deviation of normal in a subject, towithin about at least one-half standard deviation of normal, or towithin about at least one-quarter standard deviation of normal.

When an increase in the level of one or more biomarkers of mitochondrialphysiology is desired to normalize one or more biomarkers ofmitochondrial physiology, the level of the one or more biomarkers ofmitochondrial physiology can be increased to within about at least twostandard deviations of normal in a subject, increased to within about atleast one standard deviation of normal in a subject, increased to withinabout at least one-half standard deviation of normal, or increased towithin about at least one-quarter standard deviation of normal, byadministration of one or more compounds according to the presenttechnology. Alternatively, the level of one or more of the biomarkers ofmitochondrial physiology can be increased by about at least 15% abovethe subject's level of the respective one or more biomarkers ofmitochondrial physiology before administration; by about at least 20%above the subject's level of the respective one or more biomarkers ofmitochondrial physiology before administration, by about at least 30%above the subject's level of the respective one or more biomarkers ofmitochondrial physiology before administration, by about at least 40%above the subject's level of the respective one or more biomarkers ofmitochondrial physiology before administration, by about at least 50%above the subject's level of the respective one or more biomarkers ofmitochondrial physiology before administration, by about at least 75%above the subject's level of the respective one or more biomarkers ofmitochondrial physiology before administration, or by about at least100% above the subject's level of the respective one or more biomarkersof mitochondrial physiology before administration.

When a decrease in the level of one or more biomarkers of mitochondrialphysiology is desired to normalize one or more biomarkers ofmitochondrial physiology, the level of the one or more biomarkers ofmitochondrial physiology can be decreased to a level within about atleast two standard deviations of normal in a subject, decreased towithin about at least one standard deviation of normal in a subject,decreased to within about at least one-half standard deviation ofnormal, or decreased to within about at least one-quarter standarddeviation of normal, by administration of one or more compoundsaccording to the present technology. Alternatively, the level of the oneor more biomarkers of mitochondrial physiology can be decreased by aboutat least 15% below the subject's level of the respective one or morebiomarkers of mitochondrial physiology before administration, by aboutat least 20% below the subject's level of the respective one or morebiomarkers of mitochondrial physiology before administration, by aboutat least 30% below the subject's level of the respective one or morebiomarkers of mitochondrial physiology before administration, by aboutat least 40% below the subject's level of the respective one or morebiomarkers of mitochondrial physiology before administration, by aboutat least 50% below the subject's level of the respective one or morebiomarkers of mitochondrial physiology before administration, by aboutat least 75% below the subject's level of the respective one or morebiomarkers of mitochondrial physiology before administration, or byabout at least 90% below the subject's level of the respective one ormore biomarkers of mitochondrial physiology before administration.

Enhancement of the level of one or more biomarkers of mitochondrialphysiology is defined as changing the extant levels of one or morebiomarkers of mitochondrial physiology in a subject to a level whichprovides beneficial or desired effects for the subject. Sometimesnormalization of biomarkers of mitochondrial physiology may not achievethe optimum state for a subject with a disease or conditioncharacterized by mitochondrial dysfunction, and such subjects can alsobenefit from enhancement of biomarkers of mitochondrial physiology. FIG.2 illustrates the effect that various dysfunctions can have onbiochemistry and biomarkers of mitochondrial physiology.

V Monitoring Disease Progression and Therapeutic Efficacy

The methods of the present technology are useful in assessing the statusof treatment or suppression of a disease or condition characterized bymitochondrial dysfunction. For the purposes of monocyte screening, anyone or more of the biomarkers of mitochondrial physiology describedherein provide conveniently measurable benchmarks by which to gauge theeffectiveness of treatment or suppressive therapy. Additionally, otherindicators of mitochondrial physiology are known to those skilled in theart and can be monitored to evaluate the efficacy of treatment orsuppressive therapy via the methods of monocyte screening describedherein.

In some embodiments, the disclosure provides a method for evaluating theefficacy of both prophylactic and therapeutic regimens of a subjecthaving or suspected of having a disease or condition characterized bymitochondrial dysfunction via monocyte screening. For example, in someembodiments, the disclosure provides a method for evaluating theefficacy of both prophylactic and therapeutic regimens of a subjecthaving a disruption in oxidative phosphorylation. In some embodiments,monocytes are assayed via high throughput bioenergetics platforms inorder to monitor the treatment of subjects that either have or arepredisposed to having a disease or condition characterized bymitochondrial dysfunction. In some embodiments, these high throughputbioenergetics platforms permit the testing of multiple conditions perassay well. In certain embodiments, the high throughput bioenergeticsplatforms are capable of detecting the synergistic interaction of two ormore co-administered compounds.

In some embodiments, a course of treatment is recommended based on theobserved alterations in levels and/or biomarkers of mitochondrialphysiology of monocytes in a subject having or suspected of having adisease or condition characterized by mitochondrial dysfunction. In someembodiments, the course of treatment is recommended prior toadministration of a therapeutic agent, such as an aromatic-cationicpeptide of the present technology. In some embodiments, the course oftreatment is recommended after administration of a therapeutic agent,such as an aromatic-cationic peptide of the present technology. In someembodiments, the course of treatment is recommended during theadministration of a therapeutic agent, such as an aromatic-cationicpeptide of the present technology.

The methods described herein are also particularly useful to monitorprogression of a disease or condition characterized by mitochondrialdysfunction. This is because the physiological signature of circulatingmonocytes retains plasticity. Monocytes will exhibit alterations inbiomarkers of mitochondrial physiology when exposed to an inflammatorystimulus in the body (e.g., ischemia, stroke, renal injury,atherosclerosis, metabolic syndrome, acute myocardial infarction, heartfailure, ischemia reperfusion, ureteral obstruction, diabeticnephropathy, diabetes, Leber's Heredity Optic Neuropathy (LHON),Dominant Optic Atrophy (DOA), Barth's syndrome, POLG disease, Leigh'sdisease, and neurodegenerative diseases such as Alzheimer's disease,Amyotrophic Lateral Sclerosis (ALS), Friedreich's ataxia, Huntington'sdisease or Multiple Sclerosis). However, elimination of the stimuluswill cause monocytes to revert their physiological profile to baselinevalues, and vice versa when the stimulus is reintroduced. Thus,according to particular embodiments, the methods provided herein areused for monitoring the progression or recurrence of a disease orcondition characterized by mitochondrial dysfunction. This isparticularly envisaged for monitoring subjects who are at high risk ofrecurrence (for example, patients that are genetically predisposed toLHON or have a history of heart failure or stroke).

In certain embodiments, the methods described herein are useful inidentifying patient populations that exhibit different degrees ofsensitivities to a therapeutic agent (e.g., an aromatic-cationicpeptide). Age, gender, height, weight, ethnicity, family history ofgenetic disorders, immunocompromised status, and medical history arenon-limiting examples of factors that can impact responsiveness of apatient to a particular therapeutic agent. Alterations in levels and/orbiomarkers of mitochondrial physiology of monocytes can be used toclassify patients based on their responsiveness to a specific dose of atherapeutic agent (e.g., an aromatic-cationic peptide). In someembodiments, a patient may be responsive, non-responsive, orhyper-responsive to a therapeutic agent (e.g., an aromatic-cationicpeptide) at a specific dose or a range of doses. In some embodiments,monocytes are assayed via high throughput bioenergetics platforms inorder to identify responders, non-responders and hyper-responders to atherapeutic agent (e.g., an aromatic-cationic peptide).

Determining patient sensitivity to a therapeutic agent (e.g., anaromatic-cationic peptide) is useful in optimizing therapeutic efficacyand reducing side effects associated with the therapeutic agent. Incertain embodiments, the dose of the therapeutic agent (e.g., anaromatic-cationic peptide) may be adjusted to achieve therapeuticefficacy and/or minimize side effects based on alterations in levelsand/or biomarkers of mitochondrial physiology of monocytes in treatedpatients. In other embodiments, a therapeutic agent (e.g., anaromatic-cationic peptide) may be supplemented with an additionaltherapeutic agent to achieve therapeutic efficacy and/or minimize sideeffects based on alterations in levels and/or biomarkers ofmitochondrial physiology of monocytes in treated patients. In anotherembodiment, treatment with a therapeutic agent (e.g., anaromatic-cationic peptide) may be temporarily or completely discontinuedto achieve therapeutic efficacy and/or minimize side effects based onalterations in levels and/or biomarkers of mitochondrial physiology ofmonocytes in treated patients.

VI Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with an active agent such as an aromatic-cationic peptide of thepresent technology may be employed. Suitable methods include in vitro,ex vivo, or in vivo methods.

In vitro methods typically include cultured samples. For example, a cellcan be placed in a reservoir (e.g., tissue culture plate), and incubatedwith a compound under appropriate conditions suitable for obtaining thedesired result. Suitable incubation conditions can be readily determinedby those skilled in the art.

Ex vivo methods typically include cells, organs or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the compound under appropriate conditions.The contacted cells, organs or tissues are typically returned to thedonor, placed in a recipient, or stored for future use. Thus, thecompound is generally in a pharmaceutically acceptable carrier.

In vivo methods typically include the administration of an active agentsuch as an aromatic-cationic peptide of the present technology, such asthose described above, to a mammal, suitably a human. When used in vivofor therapy, the active agent such as an aromatic-cationic peptide ofthe present technology may be administered to the subject in effectiveamounts (i.e., amounts that have desired therapeutic effect). The doseand dosage regimen will depend upon the degree of the infection in thesubject, the characteristics of the particular active agent such as anaromatic-cationic peptide of the present technology used, e.g., itstherapeutic index, the subject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of an active agent such as an aromatic-cationic peptideof the present technology useful in the methods may be administered to amammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The active agent such as anaromatic-cationic peptide may be administered systemically or locally.

The aromatic-cationic peptide of the present technology may beformulated as a pharmaceutically acceptable salt. The term“pharmaceutically acceptable salt” means a salt prepared from a base oran acid which is acceptable for administration to a patient, such as amammal (e.g., salts having acceptable mammalian safety for a givendosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when an aromatic-cationic peptide of the present technology containsboth a basic moiety, such as an amine, pyridine or imidazole, and anacidic moiety such as a carboxylic acid or tetrazole, zwitterions may beformed and are included within the term “salt” as used herein. Saltsderived from pharmaceutically acceptable inorganic bases includeammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, and zinc salts, and the like.Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic,lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids(e.g., acetic, butyric, formic, propionic and trifluoroacetic acids),amino acids (e.g., aspartic and glutamic acids), aromatic carboxylicacids (e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic,hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g.,o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylicand 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylicacids (e.g., fumaric, maleic, oxalic and succinic acids), glucuronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(e.g., benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like. In someembodiments, the salt is an acetate, tartrate or trifluoroacetate salt.

The active agent such as an aromatic-cationic peptide of the presenttechnology described herein can be incorporated into pharmaceuticalcompositions for administration, singly or in combination, to a subjectfor the treatment or prevention of a medical disease or conditiondescribed herein. Such compositions typically include the active agent(e.g., an aromatic-cationic peptide) and a pharmaceutically acceptablecarrier. As used herein the term “pharmaceutically acceptable carrier”includes saline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The active agent such as an aromatic-cationic peptide of the presenttechnology can include a carrier, which can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thiomerasol, and the like. Glutathione and other antioxidants can beincluded to prevent oxidation. In many cases, isotonic agents areincluded, for example, sugars, polyalcohols such as mannitol, sorbitol,or sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, aluminummonostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the active agent such as anaromatic-cationic peptide of the present technology can be delivered inthe form of an aerosol spray from a pressurized container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer. Such methods include those described in U.S.Pat. No. 6,468,798.

Systemic administration of an active agent such as an aromatic-cationicpeptide of the present technology as described herein can also be bytransmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. In oneembodiment, transdermal administration may be performed byiontophoresis.

An active agent such as an aromatic-cationic peptide of the presenttechnology can be formulated in a carrier system. The carrier can be acolloidal system. The colloidal system can be a liposome, a phospholipidbilayer vehicle. In one embodiment, the therapeutic agent such as anaromatic-cationic peptide is encapsulated in a liposome whilemaintaining peptide integrity. One skilled in the art would appreciatethat there are a variety of methods to prepare liposomes. SeeLichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem,et al., Liposome Technology, CRC Press (1993)). Liposomal formulationscan delay clearance and increase cellular uptake (See Reddy, Ann.Pharmacother., 34(7-8):915-923 (2000)). An active agent can also beloaded into a particle prepared from pharmaceutically acceptableingredients including, but not limited to, soluble, insoluble,permeable, impermeable, biodegradable or gastroretentive polymers orliposomes. Such particles include, but are not limited to,nanoparticles, biodegradable nanoparticles, microparticles,biodegradable microparticles, nanospheres, biodegradable nanospheres,microspheres, biodegradable microspheres, capsules, emulsions,liposomes, micelles and viral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the active agent such as anaromatic-cationic peptide of the present technology can be embedded inthe polymer matrix, while maintaining protein integrity. The polymer maybe natural, such as polypeptides, proteins or polysaccharides, orsynthetic, such as poly α-hydroxy acids. Examples include carriers madeof, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulosenitrate, polysaccharide, fibrin, gelatin, and combinations thereof. Inone embodiment, the polymer is poly-lactic acid (PLA) or copolylactic/glycolic acid (PGLA). The polymeric matrices can be prepared andisolated in a variety of forms and sizes, including microspheres andnanospheres. Polymer formulations can lead to prolonged duration oftherapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923(2000)). A polymer formulation for human growth hormone (hGH) has beenused in clinical trials. See Kozarich and Rich, Chemical Biology,2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO96/40073 (Zale, et al.), and PCT publication WO 00/38651 (Shah, et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the active agents such as an aromatic-cationicpeptide of the present technology are prepared with carriers that willprotect the active agent (e.g., aromatic-cationic peptide of the presenttechnology) against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Such formulations can beprepared using known techniques. The materials can also be obtainedcommercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to specific cellswith monoclonal antibodies to cell-specific antigens) can also be usedas pharmaceutically acceptable carriers. These can be prepared accordingto methods known to those skilled in the art, for example, as describedin U.S. Pat. No. 4,522,811.

The active agent such as an aromatic-cationic peptide of the presenttechnology can also be formulated to enhance intracellular delivery. Forexample, liposomal delivery systems are known in the art, see, e.g.,Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,”Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomesfor Protein Delivery: Selecting Manufacture and Development Processes,”Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “EngineeringLiposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol.,13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996),describes the use of fusogenic liposomes to deliver a protein to cellsboth in vivo and in vitro.

Dosage, toxicity and therapeutic efficacy of the active agent such as anaromatic-cationic peptide of the present technology can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50. Insome embodiments, the active agent such as an aromatic-cationic peptideof the present technology exhibit high therapeutic indices. While anactive agent such as an aromatic-cationic peptide of the presenttechnology that exhibits toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any active agent (e.g., anaromatic-cationic peptide of the present technology) used in themethods, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to determine useful doses in humans accurately.Levels in plasma may be measured, for example, by high performanceliquid chromatography.

Typically, an effective amount of the active agent such as anaromatic-cationic peptide of the present technology, sufficient forachieving a therapeutic or prophylactic effect, ranges from about0.000001 mg per kilogram body weight per day to about 10,000 mg perkilogram body weight per day. Suitably, the dosage ranges are from about0.0001 mg per kilogram body weight per day to about 100 mg per kilogrambody weight per day. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight every day, every two days or every three days orwithin the range of 1-10 mg/kg every week, every two weeks or everythree weeks. In one embodiment, a single dosage of an active agent suchas an aromatic-cationic peptide, ranges from 0.001-10,000 micrograms perkg body weight. In one embodiment, active agent (e.g., aromatic-cationicpeptide) concentrations in a carrier range from 0.2 to 2000 microgramsper delivered milliliter. An exemplary treatment regime entailsadministration once per day or once a week. In therapeutic applications,a relatively high dosage at relatively short intervals is sometimesrequired until progression of the disease is reduced or terminated, anduntil the subject shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

In some embodiments, a therapeutically effective amount of an activeagent such as an aromatic-cationic peptide of the present technology maybe defined as a concentration of agent (e.g., peptide) at the targettissue of 10⁻¹² to 10⁻⁶ molar, e.g., approximately 10⁻⁷ molar. Thisconcentration may be delivered by systemic doses of 0.001 to 100 mg/kgor equivalent dose by body surface area. The schedule of doses would beoptimized to maintain the therapeutic concentration at the targettissue. In some embodiments, the doses are administered by single dailyor weekly administration, but may also include continuous administration(e.g., parenteral infusion or transdermal application). In someembodiments, the dosage of the active agent (e.g., aromatic-cationicpeptide of the present technology) is provided at a “low,” “mid,” or“high” dose level. In one embodiment, the low dose is provided fromabout 0.0001 to about 0.5 mg/kg/h, suitably from about 0.001 to about0.1 mg/kg/h. In one embodiment, the mid-dose is provided from about 0.01to about 1.0 mg/kg/h, suitably from about 0.01 to about 0.5 mg/kg/h. Inone embodiment, the high dose is provided from about 0.5 to about 10mg/kg/h, suitably from about 0.5 to about 2 mg/kg/h.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the medical disease or condition,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In some embodiments, the mammal is a human.

VII Evaluating the Efficacy of Combination Therapy with anAromatic-Cationic Peptide of the Present Technology and OtherTherapeutic Agents

In some embodiments, the methods of the present technology are capableof detecting the synergistic interaction of two or more co-administeredcompounds (e.g., an aromatic-cationic peptide and an additionaltherapeutic agent).

In one embodiment, an additional therapeutic agent is administered to asubject in combination with an aromatic-cationic peptide of the presenttechnology, such that a synergistic therapeutic effect is produced. A“synergistic therapeutic effect” refers to a greater-than-additivetherapeutic effect which is produced by a combination of two therapeuticagents, and which exceeds that which would otherwise result fromindividual administration of either therapeutic agent alone. Therefore,lower doses of one or both of the therapeutic agents may be used intreating a medical disease or condition, e.g., disruptions inmitochondrial oxidative phosphorylation, resulting in increasedtherapeutic efficacy and decreased side-effects.

The multiple therapeutic agents (including, but not limited to, e.g.,aromatic-cationic peptide of the present technology) may be administeredin any order or even simultaneously. If simultaneously, the multipletherapeutic agents may be provided in a single, unified form, or inmultiple forms (by way of example only, either as a single pill or astwo separate pills). One of the therapeutic agents may be given inmultiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way. For each of theexamples below, any aromatic-cationic peptide described herein could beused. By way of example, but not by limitation, the aromatic-cationicpeptide used in the examples below could be 2′6′-Dmt-D-Arg-Phe-Lys-NH₂,Phe-D-Arg-Phe-Lys-NH₂, D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or any one or more ofthe peptides shown in Section II.

Example 1: Diagnosing Transient Ischemic Stroke in a Subject UsingMonocyte Screening

This Example will demonstrate the use of monocyte screening indiagnosing transient ischemic stroke events in a subject.

Fifty patients with a reported history of transient ischemic stroke andten age-matched non-stroke controls will be selected for a clinicalstudy. Exclusion criteria will include concurrent infection, tumorbearing state, history of chronic inflammatory disease, use of drugssuch as antibiotics, immunosuppressants, and steroids within thepreceding 3 months. Blood samples are obtained from all patients afterobtaining informed consent.

The stroke and control patients will be repeatedly tested for theproportions of monocyte subsets in blood samples over a thirty dayperiod. Ethylenediaminetetraacetic acid (EDTA)-anticoagulated venousblood samples are collected from the subjects prior to each analysis.After separating mononuclear cells, monocytes are stained usingfluorescein isothiocyanate (FITC)-labelled anti-CD16 andphycoerythrin/cyanin 7 (PE-Cy7)-conjugated anti-CD14 monoclonalantibodies (BD Biosciences, San Jose, USA), and then analyzed with a BDFACSCanto flow cytometer (BD Biosciences, San Jose, USA). Determinationof CD14 and CD16 positivity will be made using the appropriate isotypecontrol antibody in keeping with the current consensus. Monocyte subsetsare defined as CD14^(high)CD16⁻ classical, CD14^(high)CD16⁺intermediate, and CD14^(low)CD16^(high) non-classical monocytes.Alterations in the biomarkers of mitochondrial physiology of theseisolated monocytes will be subsequently assessed using an XF^(e)Analyzer (Seahorse Biosciences). Patients that show elevated counts ofclassical and intermediate monocytes will be subjected to ultrasoundscans and Doppler-flow studies.

It is anticipated that the rate of transient ischemic attacks will besignificantly higher in stroke patients with elevated counts ofclassical and intermediate monocytes compared to stroke patients with areduced count of classical and intermediate monocytes. It is alsoanticipated that the overall monocyte count of the stroke group will behigher compared to that observed in the control group. Additionally, thestroke patients are expected to show an increase in disruption ofoxidative phosphorylation (as well as other abnormalities in thebiomarkers of mitochondrial physiology) compared to the age-matchedcontrols.

These results will show that methods that evaluate the overall count,distribution and biomarkers of mitochondrial physiology of monocytes areuseful as methods for diagnosing transient ischemic stroke events in asubject. The results will show that the methods described herein aregenerally useful in diagnosing a subject as having a disease orcondition characterized by mitochondrial dysfunction.

Example 2: Determining Therapeutic Efficacy of Aromatic-CationicPeptides in a Mouse Model of Huntington's Disease Using MonocyteScreening

This Example will show that monocyte screening methods of the presenttechnology can be used to determine the therapeutic efficacy ofaromatic-cationic peptides in reducing neurological defects associatedwith Huntington's Disease (HD).

R6/2 mice, expressing exon 1 of the human HD gene carrying more than 120CAG repeats, exhibit progressive neurological phenotypes that mimic thefeatures of HD in humans. The mice develop progressive neurologicalphenotypes gradually with mild phenotype (e.g., resting tremor) as earlyas 5 weeks of age and severe symptoms (including reduced mobility andseizures) at 9-11 weeks, with many of the mice dying by 14 weeks.

R6/2 HD transgenic mice are treated with an empty vehicle; or anaromatic-cationic peptide, using Alzet osmotic mini-pumps (delivering 3mg/kg/day) from age 5 weeks to 13 weeks. Monocytes are isolated fromblood samples that are drawn every third day during the 5-14 week stage.The biomarkers of mitochondrial physiology of the isolated monocyteswill be assessed by simultaneously measuring the basal oxygenconsumption, glycolysis rates, ATP production, and respiratory capacityvia the XF technology platform (Seahorse Biosciences). The R6/2 HDtransgenic animals will also be subjected to a number of behavioralassessments to study motor and cognitive function. Rotor-rod andmobility in an activity chamber are used for assessment of motorfunction, and the Y-maze is used for assessment of working memory.

Results—

It is anticipated that vehicle-treated R6/2 mice will show disruption inoxidative phosphorylation along with major motor deficits. It is furtheranticipated that treatment with the aromatic-cationic peptide willreduce the disruption in oxidative phosphorylation in isolatedmonocytes, while simultaneously improving motor activity and cognitivefunction in the R6/2 mice (as demonstrated by the animals' performancein the Y-maze test).

These results will show that methods that evaluate the biomarkers ofmitochondrial physiology of monocytes are useful as methods fordetermining the therapeutic efficacy of aromatic-cationic peptides inreducing neurological defects of HD. The results will show that themethods described herein are generally useful in evaluating the efficacyof therapeutic agents on a disease or condition characterized bymitochondrial dysfunction.

Example 3: Determining Disease Progression and Therapeutic Efficacy ofAromatic-cationic Peptides in Huntington's Disease Patients UsingMonocyte Screening

This Example will show that monocyte screening methods of the presenttechnology can be used to monitor disease progression and determine thetherapeutic efficacy of aromatic-cationic peptides in reducingneurological defects associated with Huntington's Disease (HD) in humanpatients.

HD patients exhibit progressive neurological phenotypes over timeranging from mild phenotypes (e.g., resting tremor) to extremeneurological symptoms (including reduced mobility and seizures).

Patients diagnosed with mid-stage HD are treated with an empty vehicle;or an aromatic-cationic peptide at a concentration of 10 mg/kg/day, fora period of 2 months. Monocytes are isolated from blood samples that aredrawn every third day during the 2 month treatment period. Thebiomarkers of mitochondrial physiology of the isolated monocytes will beassessed by simultaneously measuring the basal oxygen consumption,glycolysis rates, ATP production, and respiratory capacity via the XFtechnology platform (Seahorse Biosciences).

Results—

It is anticipated that vehicle-treated HD patients will show an increasein the disruption in oxidative phosphorylation in monocytes whilesimultaneously experiencing an exacerbation of neurological deficitsover the 2 month period. It is further anticipated that patients treatedwith the aromatic-cationic peptide will show a reduction in thedisruption in oxidative phosphorylation in isolated monocytes, and aconcomitant improvement in motor activity.

These results will show that methods that evaluate the biomarkers ofmitochondrial physiology of monocytes are useful as methods fordetermining the therapeutic efficacy of aromatic-cationic peptides inreducing neurological defects of HD. These methods are also useful inmonitoring the progression of HD. The results will show that the methodsdescribed herein are generally useful in evaluating the efficacy oftherapeutic agents on a disease or condition characterized bymitochondrial dysfunction and monitoring disease progression.

Example 4: Determining Therapeutic Efficacy of Aromatic-CationicPeptides in Subjects with Chronic Heart Failure Using Monocyte Screening

This example will show that monocyte screening methods of the presenttechnology can be used to determine the therapeutic efficacy ofaromatic-cationic peptides in treating chronic heart failure in a dogmodel.

Peripheral blood samples were obtained from 6 normal dogs and 6 dogswith coronary microembolizations-induced heart failure (LV ejectionfraction ˜30%). Monocytes were isolated from the peripheral bloodsamples via sequential Ficoll and Percoll density gradients. Monocyteviability, assayed using Trypan blue exclusion, was ˜70%. An XFe/XF96analyzer (Seahorse Bioscience) was used to measure oxygen consumptionrates (OCR) in monocytes in the presence and absence of 1 μM oligomycin,0.5 μM FCCP, or 1 μM each rotenone and antimycin. Mitochondrial protonleak, maximal respiration (MAXresp) and spare respiratory capacity (SRC)were measured in the presence and absence of 0.1, 1.0 and 10 μMconcentrations of D-Arg-2′6′Dmt-Lys-Phe-NH₂. Results were expressed inpmols OCR/min/μm protein.

As shown in Table 8, proton leak, MAXresp and SRC were abnormal in thecirculating monocytes of heart failure dogs compared to normal dogs.Incubation with D-Arg-2′6′Dmt-Lys-Phe-NH₂ had no effect on any of themeasures of monocyte mitochondrial function of normal dogs. In contrast,the monocyte mitochondrial function of heart failure dogs was nearlynormalized/restored after treatment with D-Arg-2′6′Dmt-Lys-Phe-NH₂, asevidenced by the dose-dependent increase in MAXresp and SRC, anddose-dependent decrease in proton leak (Table 8). These results areconsistent with the time course experiments shown in FIG. 1.

TABLE 8 Monocytes from Normal Dogs Monocytes from Heart Failure DogsPeptide concentration 0.0 μM 0.1 μM 1.0 μM 10 μM 0.0 μM 0.1 μM 1.0 μM 10μM Proton Leak (pmols 0.49 ± 0.04 0.54 ± 0.22 0.47 ± 0.06 0.48 ± 0.032.06 ± 0.23 1.55 ± 0.20* 0.94 ± 0.12* 0.77 ± 0.04* OCR/min/μg protein)MAXresp (pmols 11.4 ± 0.62 11.1 ± 0.74 12.1 ± 0.84 12.1 ± 0.67  5.5 ±0.14 6.3 ± 0.23  8.3 ± 0.20*  9.4 ± 0.78* OCR/min/μg protein) SRC (pmols6.75 ± 0.55 6.35 ± 0.64 7.04 ± 0.92 7.04 ± 0.58 1.42 ± 0.14 1.95 ± 0.20 3.64 ± 0.36* 4.51 ± 0.70* OCR/min/μg protein) *= p < 0.05 vs. 0.0 μMD-Arg-2′6′Dmt-Lys-Phe-NH₂

These results support the use of circulating monocytes as means ofassessing the therapeutic efficacy of aromatic-cationic peptides of thepresent technology in treating chronic heart failure. These methods arealso useful in monitoring the progression of chronic heart failure. Theresults show that the methods described herein are generally useful inevaluating the efficacy of therapeutic agents such as aromatic-cationicpeptides on a disease or condition characterized by mitochondrialdysfunction.

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presenttechnology is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this present technology is notlimited to particular methods, reagents, compounds compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for evaluating the therapeutic efficacyof an aromatic-cationic peptide on a disease or condition characterizedby mitochondrial dysfunction in a subject, comprising: assaying thelevel of a population of activated monocytes present in a biologicalsample obtained from the subject; and comparing the level of thepopulation of activated monocytes observed in step (a) with the level ofa corresponding population of activated monocytes observed in abiological sample obtained from the subject following administration ofa dose of an aromatic-cationic peptide, wherein the aromatic-cationicpeptide is identified as having a therapeutic effect on the disease orcondition characterized by mitochondrial dysfunction if the level of thepopulation of activated monocytes in the biological sample following theadministration of the aromatic-cationic peptide is reduced compared tothe level of the population of activated monocytes observed in step (a).2. The method of claim 1, wherein the aromatic-cationic peptide isPhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 1, wherein the level ofclassical monocytes (CD14^(high) CD16⁻) in the biological samplefollowing the administration of the aromatic-cationic peptide is reducedcompared to the level of classical monocytes observed in step (a). 4.The method of claim 1, wherein the level of intermediate monocytes(CD14^(high)CD16⁺) in the biological sample following the administrationof the aromatic-cationic peptide is reduced compared to the level ofintermediate monocytes observed in step (a).
 5. The method of claim 1,wherein the level of non-classical monocytes (CD14^(low)CD16^(high)) inthe biological sample following the administration of thearomatic-cationic peptide is increased compared to the level ofnon-classical monocytes observed in step (a).
 6. The method of claim 1,wherein the disease or condition characterized by mitochondrialdysfunction is ischemia, stroke, renal injury, neurodegenerativedisease, atherosclerosis, metabolic syndrome, acute myocardialinfarction, heart failure, ischemia reperfusion, ureteral obstruction,diabetic nephropathy, diabetes, Leber's Heredity Optic Neuropathy(LHON), Dominant Optic Atrophy (DOA), Barth's syndrome, POLG disease, orLeigh's disease.
 7. The method of claim 6, wherein the neurodegenerativedisease is Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS),Huntington's disease, Friedreich's ataxia or Multiple Sclerosis.
 8. Amethod for evaluating the therapeutic efficacy of an aromatic-cationicpeptide on a disease or condition characterized by mitochondrialdysfunction in a subject, comprising: a. assaying the ratio of differentmonocyte types present in a biological sample obtained from the subject;and b. comparing the ratio of different monocyte types observed in step(a) with the ratio of corresponding monocyte types observed in abiological sample obtained from the subject following administration ofa dose of an aromatic-cationic peptide, wherein the aromatic-cationicpeptide is identified as having a therapeutic effect on the disease orcondition characterized by mitochondrial dysfunction if the ratio ofdifferent monocyte types in the biological sample following theadministration of the aromatic-cationic peptide is altered compared tothe ratio of different monocyte types observed in step (a).
 9. Themethod of claim 8, wherein the ratio of classical monocytes tonon-classical monocytes in the biological sample following theadministration of the aromatic-cationic peptide is reduced compared tothe ratio of classical monocytes to non-classical monocytes observed instep (a).
 10. The method of claim 8, wherein the ratio of intermediatemonocytes to non-classical monocytes in the biological sample followingthe administration of the aromatic-cationic peptide is reduced comparedto the ratio of intermediate monocytes to non-classical monocytesobserved in step (a).
 11. The method of claim 8, wherein the ratio ofclassical monocytes to intermediate monocytes in the biological samplefollowing the administration of the aromatic-cationic peptide is reducedcompared to the ratio of classical monocytes to intermediate monocytesobserved in step (a).
 12. The method of claim 8, wherein the disease orcondition characterized by mitochondrial dysfunction is ischemia,stroke, renal injury, neurodegenerative disease, atherosclerosis,metabolic syndrome, acute myocardial infarction, heart failure, ischemiareperfusion, ureteral obstruction, diabetic nephropathy, diabetes,Leber's Heredity Optic Neuropathy (LHON), Dominant Optic Atrophy (DOA),Barth's syndrome, POLG disease, or Leigh's disease.
 13. The method ofclaim 12, wherein the neurodegenerative disease is Alzheimer's disease,Amyotrophic Lateral Sclerosis (ALS), Huntington's disease, Friedreich'sataxia or Multiple Sclerosis.
 14. A method for evaluating thetherapeutic efficacy of an aromatic-cationic peptide on a disease orcondition characterized by mitochondrial dysfunction in a subject,comprising: a. assaying at least one biomarker of mitochondrialphysiology of a population of monocytes present in a biological sampleobtained from the subject; and b. comparing the biomarker ofmitochondrial physiology of the population of monocytes observed in step(a) with the biomarker of mitochondrial physiology of a correspondingpopulation of monocytes observed in a biological sample obtained fromthe subject following administration of a dose of an aromatic-cationicpeptide, wherein the aromatic-cationic peptide is identified as having atherapeutic effect on the disease or condition characterized bymitochondrial dysfunction if the biomarker of mitochondrial physiologyof the population of monocytes in the biological sample following theadministration of the aromatic-cationic peptide is similar to thebiomarker of mitochondrial physiology of a corresponding population ofmonocytes in a reference sample obtained from a healthy subject.
 15. Themethod of claim 14, wherein the aromatic-cationic peptide isPhe-D-Arg-Phe-Lys-NH₂ or D-Arg-2′6′Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof.
 16. The method of claim 14, wherein thealterations in biomarkers of mitochondrial physiology is determinedusing assays that measure disruption in oxidative phosphorylation. 17.The method of claim 16, wherein the disruption in oxidativephosphorylation is determined using assays that measure CoQ10 levels,uncoupling ratio, net routine flux control ratio, leak flux controlratio or phosphorylation respiratory control ratio.
 18. The method ofclaim 14, wherein the alterations in biomarkers of mitochondrialphysiology of monocytes is determined by measuring alterations in thelevel of one or more biomarkers of mitochondrial physiology in a sampleof monocytes.
 19. The method of claim 18, wherein the biomarkers ofmitochondrial physiology are selected from the group consisting ofconsisting of lactic acid (lactate) levels, pyruvic acid (pyruvate)levels, lactate/pyruvate ratios, phosphocreatine levels, NADH (NADH+H³⁰)or NADPH (NADPH+H³⁰) levels; NAD or NADP levels; ATP levels; reducedcoenzyme Q (CoQred) levels; oxidized coenzyme Q (CoQox) levels; totalcoenzyme Q (CoQtot) levels; oxidized cytochrome C levels; reducedcytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio;acetoacetate levels; beta-hydroxy butyrate levels;acetoacetate/beta-hydroxy butyrate ratio; 8-hydroxy-2′-deoxyguanosine(8-OHdG) levels; levels of reactive oxygen species; oxygen consumption(VO2), carbon dioxide output (VCO2), and respiratory quotient(VCO2/VO2).
 20. The method of claim 14, wherein alterations in thebiomarkers of mitochondrial physiology are determined using highthroughput bioenergetics platforms.